Eflornithine and sulindac, fixed dose combination formulation

ABSTRACT

Provided herein are fixed-dose combination formulations of a pharmaceutically effective amount of eflornithine together with a pharmaceutically effective amount of sulindac. Also provided are methods of use and of methods of manufacture of these formulations.

The present application is a continuation of U.S. application Ser. No.15/771,484, filed Apr. 27, 2018, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2016/059689, filed Oct. 31, 2016, which claims the prioritybenefit of U.S. provisional application No. 62/248,810, filed Oct. 30,2015, U.S. provisional application No. 62/358,698, filed Jul. 6, 2016,European application No. 16306429.8, filed Oct. 28, 2016, and Europeanapplication No. 16306430.6, filed Oct. 28, 2016, the entire contents ofeach of which are specifically incorporated herein by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of cancer biologyand medicine. More particularly, it concerns compositions for theprevention and treatment of carcinomas.

2. Description of Related Art

Cancer cells have the ability to co-opt multiple pathways to fulfilltheir increased requirement for specific metabolites (Vander Heiden,2011). The nonsteroidal anti-inflammatory drugs (NSAIDs), includingaspirin, ibuprofen, piroxicam (Reddy et al., 1990; Singh et al., 1994),indomethacin (Narisawa, 1981), and sulindac (Piazza et al., 1997; Rao etal., 1995), effectively inhibit colon carcinogenesis in the AOM-treatedrat model. Sulindac sulfone, a metabolite of the NSAID sulindac, lacksCOX-inhibitory activity yet induces apoptosis in tumor cells (Piazza etal., 1995; Piazza et al., 1997b) and inhibits tumor development inseveral rodent models of carcinogenesis (Thompson et al., 1995; Piazzaet al., 1995, 1997a).

α-Difluoromethylornithine (DFMO) is an enzyme-activated, irreversibleinhibitor of ornithine decarboxylase (ODC) and causes depletion in theintracellular concentrations of putrescine and its derivative,spermidine (Pegg, 1988). In experimental animal models, DFMO is a potentinhibitor of carcinogenesis that is especially active in preventingcarcinogen-induced epithelial cancers of many organs, including those ofthe colon (Weeks et al., 1982; Thompson et al., 1985; Nowels et al.,1986; Nigro et al., 1987).

A major impediment to the translation of cancer chemoprevention researchinto clinical practice has been marginal agent efficacy and toxicitiesthat exceed benefit (Psaty and Potter, 2006; Lippman, 2006). Forexample, the demonstrated marked efficacy of polyamine-inhibitorycombination of long-term daily oral D,L-α-difluoromethylornithine (DFMO,eflornithine) and sulindac among colorectal adenoma (CRA) patients hasbeen demonstrated (Meyskens et al., 2008), however, treatment wasassociated with modest, subclinical ototoxicity (McLaren et al., 2008),and a greater number of cardiovascular events among patients with highbaseline cardiovascular risk (Zell et al., 2009).

The convenience of co-administering two or more active pharmaceuticalingredients in a unit dosage form, as opposed to the administration of anumber of separate doses of two or more pharmaceuticals at regularintervals, has been recognized in the pharmaceutical arts and isdescribed in U.S. Pat. Nos. 6,428,809 and 6,702,683. Potentialadvantages to the patient and clinician include (1) minimization orelimination of local and/or systemic side effects; (2) more effectivetreatment of co-morbid conditions; (3) improved polypharmacy; and (4)better patient compliance with overall disease management, which in turnmay lead to reduced costs due to fewer trips to the physician, reducedhospitalization, and improved patient well-being. Fixed dose combinationproducts, with two or more formulations combined or co-formulated in asingle dosage form, may be useful in multiple drug regimens whereimproved clinical effectiveness, enhanced patient adherence andsimplified dosing are desired. However, pharmaceutical drug productdevelopment of solid oral dosage forms is complicated at both theresearch and development level and at the commercial manufacturing leveleven for single active pharmaceutical ingredient (API) formulation. Formore-than-one API, additional complicating factors are expected,including (1) drug-drug interaction, (2) drug-excipient interaction, (3)simultaneous release profiles, (4) differential release profiles, and(5) blend uniformity of each drug component. In view of these hurdles,developing fixed-dose combinations with the same or similar releaseprofiles as the single entity drug products typically represents asignificant challenge. Fixed dose combinations of eflornithine andsulindac that overcome some or all of these challenges would have asignificant potential impact for the effective treatment and/orprevention of a wide range of diseases or disorders, including familialadenoma polyposis (FAP).

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compositions comprising afixed dose combination of a pharmaceutically effective amount ofeflornithine and a pharmaceutically effective amount of a nonsteroidalanti-inflammatory drug (NSAID) or a metabolite thereof. In someembodiments, the fixed dose combination is a pharmaceutically effectiveamount of eflornithine and a pharmaceutically effective amount ofsulindac.

In some embodiments, the eflornithine is eflornithine hydrochloridemonohydrate. In some embodiments, the eflornithine is eflornithinehydrochloride monohydrate racemate. In some embodiments, theeflornithine hydrochloride monohydrate is a racemic mixture of its twoenantiomers. In some embodiments, the eflornithine hydrochloridemonohydrate is a substantially optically pure preparation. In someembodiments, the eflornithine hydrochloride monohydrate isL-eflornithine hydrochloride monohydrate or D-eflornithine hydrochloridemonohydrate. In some embodiments, the eflornithine is anhydrous freebase eflornithine.

In some embodiments, the eflornithine is present in an amount of about10 to about 1000 mg. In some embodiments, the eflornithine is present inan amount of about 250 to about 500 mg. In some embodiments, theeflornithine is present in an amount of about 300 to about 450 mg. Insome embodiments, the eflornithine is present in an amount of about 350to about 400 mg. In some embodiments, the eflornithine is present in anamount of about 35 to about 60 weight percent. In some embodiments, theeflornithine is present in an amount of about 40 to about 55 weightpercent. In some embodiments, the eflornithine is present in an amountof about 50 to about 55 weight percent. In some embodiments, theeflornithine is present in an amount of about 52 to about 54 weightpercent. In some embodiments, the amount of eflornithine hydrochloridemonohydrate racemate is from 52 to 54 weight percent. In someembodiments, the eflornithine is present in an amount of about 375 mg.In some embodiments, the amount of eflornithine hydrochloridemonohydrate racemate is 375 mg.

In some embodiments, the sulindac is present in an amount from about 10to about 1500 mg. In some embodiments, the sulindac is present in anamount of about 50 to about 100 mg. In some embodiments, the sulindac ispresent in an amount of about 70 to about 80 mg. In some embodiments,the sulindac is present in an amount of about 75 mg. In someembodiments, the amount of sulindac is 75 mg. In some embodiments, thesulindac is present in an amount of about 5 to about 20 weight percent.In some embodiments, the sulindac is present in an amount of about 8 toabout 15 weight percent. In some embodiments, the sulindac is present inan amount of about 10 to about 12 weight percent. In some embodiments,the amount of sulindac is from 10 to 11 weight percent.

In some embodiments, the eflornithine is present in an amount of about375 mg and the sulindac is present in an amount of about 75 mg.

In some embodiments, the formulation further comprises an excipient. Insome embodiments, the excipient is starch, colloidal silicon dioxide, orsilicified microcrystalline cellulose. In some embodiments, theexcipient is colloidal silicon dioxide. In some embodiments, theformulation further comprises a second excipient. In some embodiments,the second excipient is silicified microcrystalline cellulose.

In some embodiments, the formulation further comprises a lubricant. Insome embodiments, the lubricant is magnesium stearate, calcium stearate,sodium stearate, glyceryl monostearate, aluminum stearate, polyethyleneglycol, boric acid or sodium benzoate. In some embodiments, thelubricant is magnesium stearate. In some embodiments, magnesium stearateis present in an amount of about 0.25 to about 2 weight percent. In someembodiments, the amount of magnesium stearate is from about 0.75 toabout 2 weight percent. In some embodiments, the amount of magnesiumstearate is from about 1 to about 1.5 weight percent. In someembodiments, the amount of magnesium stearate is about 1.1 weightpercent. In some embodiments, magnesium stearate is present in an amountof about 1.5 weight percent.

In some embodiments, the compositions are in the form of a capsule,tablet, mini tablets, granules, pellets, solution, gel, cream, foam orpatch. In some embodiments, the composition is in the form of a tablet,for example, a monolayer tablet.

In some embodiments, the weight of the tablet is from about 10 mg toabout 2,500 mg. In some embodiments, the weight of the tablet is fromabout 250 mg to about 1,500 mg. In some embodiments, the weight of thetablet is from about 650 mg to about 1,000 mg. In some embodiments, theweight of the tablet is from about 675 mg to about 725 mg. In someembodiments, the weight of the tablet is about 700 mg.

In some embodiments, the weight of the capsule, mini tablet, granules,or pellets is from about 10 mg to about 2,500 mg. In some embodiments,the weight of the capsule, mini tablet, granules, or pellets is fromabout 250 mg to about 1,500 mg. In some embodiments, the weight of thecapsule, mini tablet, granules, or pellets is from about 650 mg to about1,000 mg. In some embodiments, the weight of the capsule, mini tablet,granules, or pellets is from about 675 mg to about 725 mg. In someembodiments, the weight of the capsule, mini tablet, granules, orpellets is about 700 mg.

In some embodiments, the tablet further comprises a coating. In someembodiments, the coating is a modified release coating or an entericcoating. In some embodiments, the coating is a pH-responsive coating. Insome embodiments, the coating comprises cellulose acetate phthalate(CAP), cellulose acetate trimelletate (CAT), poly (vinyl acetate)phthalate (PVAP), hydroxypropylmethylcellulose phthalate (HP),poly(methacrylate ethylacrylate) (1:1) copolymer (MA-EA),poly(methacrylate methylmethacrylate) (1:1) copolymer (MA MMA),poly(methacrylate methylmethacrylate) (1:2) copolymer, orhydroxypropylmethylcellulose acetate succinate (HPMCAS). In someembodiments, the coating masks the taste of eflornithine. In someembodiments, the coating comprises hydroxypropyl methylcellulose,titanium dioxide, polyethylene glycol, and iron oxide yellow.

In some embodiments, the amount of coating is from about 1 to about 5weight percent. In some embodiments, the amount of coating is from about2 to about 4 weight percent. In some embodiments, the amount of coatingis about 3 weight percent. In some embodiments, the amount of coating isfrom about 5 mg to about 30 mg. In some embodiments, the amount ofcoating is from about 15 mg to about 25 mg. In some embodiments, theamount of coating is about 21 mg.

In some embodiments, the weight of the tablet comprising a coating isfrom about 675 mg to about 750 mg. In some embodiments, the weight ofthe tablet comprising a coating is from about 700 mg to about 725 mg. Insome embodiments, the weight of the tablet comprising a coating is about721 mg.

In one aspect, there is provided a method of preventing and/or treatinga disease or condition in a patient in need thereof, comprisingadministering to the patient the fixed dose combination of apharmaceutically effective amount of eflornithine and a pharmaceuticallyeffective amount of sulindac provided herein.

In some embodiments, the method further comprises administering to thepatient a second composition comprising the fixed dose combination of apharmaceutically effective amount of eflornithine and a pharmaceuticallyeffective amount of sulindac provided herein. In some embodiments, thefirst and the second compositions comprise the same fixed dosecombinations. In some embodiments, the first and the secondadministration occurs simultaneously. In some embodiments, the secondadministration follows the first administration by an interval of 1second to 1 hour. In some embodiments, the first and the secondcompositions are both formulated as tablets and contain the same amountsof eflornithine and sulindac.

In some embodiments, the disease is cancer. In some embodiments, thecancer is colon cancer, breast cancer, pancreatic cancer, brain cancer,lung cancer, stomach cancer, a blood cancer, skin cancer, testicularcancer, prostate cancer, ovarian cancer, liver cancer, or esophagealcancer. In some embodiments, the colon cancer is familial adenomatouspolyposis. In some embodiments, the cancer is a neuroendocrine tumor. Insome embodiments, the neuroendocrine tumor is neuroblastoma.

In some embodiments, the condition is a skin condition. In someembodiments, the skin condition is facial hirsutism.

In some embodiments, the composition is administered orally,intraarterially, intravenously, or topically. In some embodiments, thecomposition is administered orally.

In some embodiments, the composition is administered orally. In someembodiments, the composition is administered every 12 hours. In someembodiments, the composition is administered every 24 hours. In someembodiments, the composition is administered at least a second time.

In another aspect, there is provided a method of producing a tabletcomprising about 375 mg eflornithine hydrochloride and about 75 mg ofsulindac comprising: (a) pre-mixing sulindac and an excipient to form afirst mixture; (b) mixing the first mixture with a second mixturecomprising eflornithine and an excipient to form a blend; (c) screeningthe blend to form a granulated blend; (d) adding a lubricant to thegranulated blend to obtain a final blend; and (e) applying a compressionforce to the final blend to form a tablet. In some embodiments, themethod further comprises mixing the granulated blend prior to step (d)and mixing the final blend prior to step (e).

In some embodiments, there are two excipients in the first mixture,wherein the first excipient is colloidal silicon dioxide, and the secondexcipient is silicified microcrystalline cellulose. In some embodiments,the excipient of the second mixture is silicified microcrystallinecellulose.

In some embodiments, the pre-mixing is performed in apolyethylene-coated container. In some embodiments, the mixing isperformed in a diffusion blender.

In some embodiments, the lubricant is magnesium stearate. In someembodiments, the magnesium stearate is sieved through a screen prior tostep (d). In some embodiments, the screen is a 500 μm screen.

In some embodiments, screening comprises applying the blend to arotative calibrator. In some embodiments, the rotative calibratorcomprises a 1.0 mm screen.

In some embodiments, the method further comprises a pre-compression stepafter step (d) and prior to step (e), wherein the blend is compressedwith a force lower than the force of step (e) to form a pre-compressedblend, further wherein the compression force of step (e) then acts onthe pre-compressed blend to form the tablet. In some embodiments, thepre-compression step prevents tablet capping. In some embodiments, acompression force of the pre-compression step is applied at about 5 toabout 15 percent of the compression force applied in step (e). In someembodiments, the compression force of the pre-compression step is from2.5 to 3.5 kN. In some embodiments, the compression force of thepre-compression step is about 3 kN. In some embodiments, the compressionforce of step (e) is from 20 to 35 kN. In some embodiments, thecompression force of step (e) is about 25 kN.

In some embodiments, the method further comprises coating the tablet. Insome embodiments, the coating comprises hydroxypropyl methylcellulose,titanium dioxide, polyethylene glycol, and iron oxide yellow.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Stability analysis of prototype Lot 7107/04 of 700 mg tablets ofeflornithine HCl monohydrate (375 mg) and sulindac (75 mg). Tablets havea 3% w/w coating. Samples were analyzed at time zero (T0) and at 6months (T6) using a validated Karl Fischer titration method fordetermination of water content. Samples were stored in HDPE bottles withand without caps in verified stability chambers. Values represent thepercentage of water in each tablet at the specified conditions.

FIGS. 2A-2B: Results of dissolution analysis of coated tablet Lots7107/04 and 6A001. Reference tablets of 250 mg eflornithine HClmonohydrate and commercial 150 mg sulindac are included for comparison.Co-formulated tablets contain 375 mg of eflornithine HCl monohydrate and75 mg of sulindac with a 3% w/w coating.

FIG. 3: Simplified scheme depicting a manufacturing process for tabletscontaining both eflornithine HCl monohydrate and sulindac.

FIGS. 4A-4C: (A) A typical HPLC chromatograph of eflornithine HClmonohydrate and sulindac co-formulated tablet demonstrating the abilityto measure selected impurities. (B-C) X-ray powder diffraction (XRPD)patterns of eflornithine HCl monohydrate and sulindac active ingredientsmixed with tablet excipients at time zero, 2 weeks, and 4 weeks. Thelack of change supports both excipient compatibility and polymorphstability.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In several aspects, compositions are provided for a fixed dosecombination (FDC) of eflornithine and sulindac. Methods are alsoprovided for the manufacture of the fixed dose combinations of thepresent invention which overcome problems associated with currentmethods. The methods of manufacture have been designed to solve problemsincluding drug-drug interactions, drug-excipient interactions, and blenduniformity of each drug component. Accordingly, the fixed dosecombination of the present invention may be used to minimize localand/or systemic side effects, provide more effective treatments, improvepolypharmacy, and provide better patient compliance.

I. FAMILIAL ADENOMATOUS POLYPOSIS

Excess polyamine formation has long been implicated in epithelialcarcinogenesis, particularly colorectal carcinogenesis. Polyamines aresmall ubiquitous molecules involved in various processes, including, forexample, transcription, RNA stabilization, and ion channel gating(Wallace, 2000). Ornithine decarboxylase (ODC), the first enzyme inpolyamine synthesis, is essential for normal development and tissuerepair in mammals but is down-regulated in most adult tissues (Gernerand Meyskens, 2004). Multiple abnormalities in the control of polyaminemetabolism and transport result in increased polyamine levels that canpromote tumorigenesis in several tissues (Thomas and Thomas, 2003).

Familial adenomatous polyposis (FAP) is a syndrome associated with highrisk of colon and other cancers. FAP is caused by mutations in theadenomatous polyposis coli (APC) tumor suppressor gene, and APCsignaling has been shown to regulate ODC expression in both human cells(Fultz and Gerner, 2002) and in a mouse model of FAP (Erdman et al.,1999). Polyamine metabolism is up-regulated in intestinal epithelialtissues of humans with (Giardiello et al., 1997) FAP.

Wild type APC expression leads to decreased expression of ODC, whilemutant APC leads to increased expression of ODC. The mechanism ofAPC-dependent regulation of ODC involves E-box transcription factors,including the transcriptional activator c-MYC and the transcriptionalrepressor MAD1 (Fultz and Gerner, 2002; Martinez et al., 2003). c-MYCwas shown by others to regulate ODC transcription (Bellofernandez etal., 1993). Several genes involved in polyamine metabolism are essentialgenes for optimal growth in most organisms, and are down-regulated innon-proliferating and/or adult cells and tissues (Gerner and Meyskens,2004). The polyamines influence specific cellular phenotypes, in part,by affecting patterns of gene expression, as reviewed elsewhere (Childset al., 2003).

Familial Adenomatous Polyposis (FAP), an inherited polyposis syndrome,is the result of germ-line mutation of the adenomatous polyposis coli(APC) tumor suppressor gene (Su et al., 1992). This autosomal-dominantcondition with variable expression is associated with the development ofhundreds of colonic adenomas, which uniformly progress to adenocarcinomaby forty years of age, two decades earlier than the mean age diagnosisfor sporadic colon cancer (Bussey, 1990). In prior studies ofpre-symptomatic individuals with FAP, increased levels of the polyaminesspermidine and spermine, and their diamine precursor putrescine, havebeen detected in normal-appearing colorectal biopsies when compared tonormal family member controls (Giardiello et al., 1997). The activity ofornithine decarboxylase (ODC), the first and rate-limiting enzyme inmammalian polyamine synthesis, also is elevated in apparently normalcolonic mucosal biopsies from FAP patients (Giardiello et al., 1997; Lukand Baylin, 1984). These findings are of interest as the polyamines arenecessary for optimal cell proliferation (Pegg, 1986). Further,suppression of ODC activity, using the enzyme-activated irreversibleinhibitor DFMO, inhibits colon carcinogenesis in carcinogen-treatedrodents (Kingsnorth et al., 1983; Tempero et al., 1989).

The Min (multiple intestinal neoplasia) mouse, which shares a mutatedAPC/apc genotype with FAP, serves as a useful experimental animal modelfor human FAP patients (Lipkin, 1997). The Min mouse can develop greaterthan 100 gastrointestinal adenomas/adenocarcinomas throughout thegastrointestinal tract by 120 days of life leading to GI bleeding,obstruction and death. A combination therapy of DFMO and sulindac wasshown to be effective in reducing adenomas in these mice. See U.S. Pat.No. 6,258,845 and Gerner and Meyskens, 2004, which are incorporatedherein by reference.

II. EFLORNITHINE

The term “eflornithine” when used by itself and free of context refersto 2,5-diamino-2-(difluoromethyl)pentanoic acid in any of its forms,including non-salt and salt forms (e.g., eflornithine HCl), anhydrousand hydrate forms of non-salt and salt forms (e.g., eflornithinehydrochloride monohydrate), solvates of non-salt and salts forms, itsenantiomers (R and S forms, which may also by identified as d and 1forms), and mixtures of these enantiomers (e.g., racemic mixture). By“substantially optically pure preparation” is meant a preparation of afirst enantiomer which contains about 5% wt. or less of the oppositeenantiomer. Specific forms of eflornithine include eflornithinehydrochloride monohydrate (i.e., CAS ID: 96020-91-6; MW: 236.65),eflornithine hydrochloride (i.e., CAS ID: 68278-23-9; MW: 218.63), andanhydrous free base eflornithine (i.e., CAS ID: 70052-12-9; MW: 182.17).Where necessary, the specific form of eflornithine has been furtherspecified. In some embodiments, the eflornithine of the presentdisclosure is eflornithine hydrochloride monohydrate (i.e., CAS ID:96020-91-6). The terms “eflornithine” and “DFMO” are usedinterchangeably herein. DFMO is an abbreviation fordifluoromethylornithine. Other synonyms of eflornithine and DFMOinclude: α-difluoromethylornithine, 2-(difluoromethyl)-DL-ornithine,2-(difluoromethyl)-dl-ornithine, 2-(Difluoromethyl)ornithine,DL-α-difluoromethylornithine, N-Difluoromethylornithine,αδ-diamino-α-(difluoromethyl)valeric acid, and2,5-diamino-2-(difluoromethyl)pentanoic acid.

Eflornithine is an enzyme-activated irreversible inhibitor of ornithinedecarboxylase (ODC), the rate-limiting enzyme of the polyaminebiosynthetic pathway. As a result of this inhibition of polyaminesynthesis, the compound is effective in preventing cancer formation inmany organ systems, inhibiting cancer growth, and reducing tumor size.It also has synergistic action with other antineoplastic agents.

Eflornithine has been shown to decrease APC-dependent intestinaltumorigenesis in mice (Erdman et al., 1999). Oral eflornithineadministered daily to humans inhibits ODC enzyme activity and polyaminecontents in a number of epithelial tissues (Love et al., 1993; Gerner etal., 1994; Meyskens et al., 1994; Meyskens et al., 1998; Simoneau etal., 2001; Simoneau et al., 2008). Eflornithine in combination with thenon-steroidal anti-inflammatory drug (NSAID) sulindac, has been reportedto markedly lower the adenoma recurrence rate among individuals withcolonic adenomas when compared to placebos in a randomized clinicaltrial (Meyskens et al., 2008).

Eflornithine was originally synthesized by Centre de Recherche Merrell,Strasbourg. Current U.S. Food and Drug Administration (FDA) approvalsinclude:

-   -   African sleeping sickness. High dose systemic IV dosage form—not        marketed (Sanofi/WHO)    -   Hirsutis (androgen-induced excess hair growth) topical dosage        form        While no oral formulations of eflornithine have yet been        approved by the FDA, topical and injectable forms have been        approved. Vaniqa® is a cream, which contains 15% w/w        eflornithine hydrochloride monohydrate, corresponding to 11.5%        w/w anhydrous eflornithine (EU), respectively 13.9% w/w        anhydrous eflornithine hydrochloride (U.S.), in a cream for        topical administration. Ornidyl® is an eflornithine HCl solution        suitable for injection or infusion. It is supplied in the        strength of 200 mg eflornithine hydrochloride monohydrate per ml        (20 g/100 mL).

Eflornithine and its use in the treatment of benign prostatichypertrophy are described in U.S. Pat. Nos. 4,413,141, and 4,330,559.The '141 Patent describes eflornithine as being a powerful inhibitor ofODC, both in vitro and in vivo. Administration of eflornithine isreported to cause a decrease in putrescine and spermidine concentrationsin cells in which these polyamines are normally actively produced.Additionally, eflornithine has been shown to be capable of slowingneoplastic cell proliferation when tested in standard tumor models. The'559 Patent describes the use of eflornithine and eflornithinederivatives for the treatment of benign prostatic hypertrophy. Benignprostatic hypertrophy, like many disease states characterized by rapidcell proliferation, is accompanied by abnormal elevation of polyamineconcentrations.

Eflornithine can potentially be given continuously with significantanti-tumor effects. This drug is relatively non-toxic at low doses of0.4 g/m²/day to humans while producing inhibition of putrescinesynthesis in tumors. Studies in a rat-tumor model demonstrate thateflornithine infusion can produce a 90% decrease in tumor putrescinelevels without suppressing peripheral platelet counts.

Side effects observed with eflornithine include effects on hearing athigh doses of 4 g/m²/day that resolve when it is discontinued. Theseeffects on hearing are not observed at lower doses of 0.4 g/m²/day whenadministered for up to one year (Meyskens et al., 1994). In addition, afew cases of dizziness/vertigo are seen that resolve when the drug isstopped. Thrombocytopenia has been reported predominantly in studiesusing high “therapeutic” doses of eflornithine (>1.0 g/m²/day) andprimarily in cancer patients who had previously undergone chemotherapyor patients with compromised bone marrow. Although the toxicityassociated with eflornithine therapy is not, in general, as severe asother types of chemotherapy, in limited clinical trials it has beenfound to promote a dose-related thrombocytopenia. Moreover, studies inrats have shown that continuous infusion of eflornithine for 12 dayssignificantly reduces platelet counts compared with controls. Otherinvestigations have made similar observations in which thrombocytopeniais the major toxicity of continuous intravenous eflornithine therapy.These findings suggest that eflornithine may significantly inhibit ODCactivity of the bone marrow precursors of megakaryocytes. Eflornithinemay inhibit proliferative repair processes, such as epithelial woundhealing.

A phase III clinical trial assessed the recurrence of adenomatous polypsafter treatment for 36 months with DFMO plus sulindac or matchedplacebos. Temporary hearing loss is a known toxicity of treatment withDFMO, thus a comprehensive approach was developed to analyze serial airconduction audiograms. The generalized estimating equation methodestimated the mean difference between treatment arms with regard tochange in air conduction pure tone thresholds while accounting forwithin-subject correlation due to repeated measurements at frequencies.Based on 290 subjects, there was an average difference of 0.50 dBbetween subjects treated with DFMO plus sulindac compared with thosetreated with placebo (95% confidence interval, −0.64 to 1.63 dB;P=0.39), adjusted for baseline values, age, and frequencies. There is a<2 dB difference in mean threshold for patients treated with DFMO plussulindac compared with those treated with placebo. The results of thisstudy are discussed in greater detail in McLaren et al., 2008, which isincorporated herein by reference in its entirety.

III. NSAIDS

NSAIDs are anti-inflammatory agents that are not steroids. In additionto anti-inflammatory effects, they are also reported to have analgesic,antipyretic, and platelet-inhibitory effects. They are used, forexample, in the treatment of chronic arthritic conditions and certainsoft tissue disorders associated with pain and inflammation. They havebeen reported to act by blocking the synthesis of prostaglandins byinhibiting cyclooxygenase, which converts arachidonic acid to cyclicendoperoxides, precursors of prostaglandins. Inhibition of prostaglandinsynthesis accounts for their analgesic, antipyretic, andplatelet-inhibitory actions; other mechanisms may contribute to theiranti-inflammatory effects. Certain NSAIDs also may inhibit lipoxygenaseenzymes or phospholipase C or may modulate T-cell function. See AMA DrugEvaluations Annual, 1814-5, 1994.

The nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin,ibuprofen, piroxicam (Reddy et al., 1990; Singh et al., 1994),indomethacin (Narisawa, 1981), and sulindac (Piazza et al., 1997; Rao etal., 1995), effectively inhibit colon carcinogenesis in the AOM-treatedrat model. NSAIDs also inhibit the development of tumors harboring anactivated Ki-ras (Singh and Reddy, 1995). NSAIDs appear to inhibitcarcinogenesis via the induction of apoptosis in tumor cells (Bedi etal., 1995; Lupulescu, 1996; Piazza et al., 1995; Piazza et al., 1997b).A number of studies suggest that the chemopreventive properties of theNSAIDs, including the induction of apoptosis, are a function of theirability to inhibit prostaglandin synthesis (reviewed in DuBois et al.,1996; Lupulescu, 1996; Vane and Botting, 1997). Studies, however,indicate that NSAIDs may act through both prostaglandin-dependent and-independent mechanisms (Alberts et al., 1995; Piazza et al., 1997a;Thompson et al., 1995; Hanif, 1996). Sulindac sulfone, a metabolite ofthe NSAID sulindac, lacks COX-inhibitory activity yet induces apoptosisin tumor cells (Piazza et al., 1995; Piazza et al., 1997b) and inhibitstumor development in several rodent models of carcinogenesis (Thompsonet al., 1995; Piazza et al., 1995, 1997a).

Several NSAIDs have been examined for their effects in human clinicaltrials. A phase IIa trial (one month) of ibuprofen was completed andeven at the dose of 300 mg/day, a significant decrease in prostoglandinE₂ (PGE₂) levels in flat mucosa was seen. A dose of 300 mg of ibuprofenis very low (therapeutic doses range from 1200-3000 mg/day or more), andtoxicity is unlikely to be seen, even over the long-term. However, inanimal chemoprevention models, ibuprofen is less effective than otherNSAIDs.

A. Aspirin

Aspirin, also known as acetylsalicylic acid, is a salicylate drug, oftenused as an analgesic to relieve minor aches and pains, as an antipyreticto reduce fever, and as an anti-inflammatory medication. Aspirin wasfirst isolated by Felix Hoffmann, a chemist with the German companyBayer in 1897. Salicylic acid, the main metabolite of aspirin, is anintegral part of human and animal metabolism. While in humans much of itis attributable to diet, a substantial part is synthesized endogenously.Today, aspirin is one of the most widely used medications in the world,with an estimated 40,000 tons of it being consumed each year. Incountries where aspirin is a registered trademark owned by Bayer, thegeneric term is acetylsalicylic acid (ASA).

Aspirin also has an antiplatelet effect by inhibiting the production ofthromboxane, which under normal circumstances binds platelet moleculestogether to create a patch over damaged walls of blood vessels. Becausethe platelet patch can become too large and also block blood flow,locally and downstream, aspirin is also used long-term, at low doses, tohelp prevent heart attacks, strokes, and blood clot formation in peopleat high risk of developing blood clots. It has also been establishedthat low doses of aspirin may be given immediately after a heart attackto reduce the risk of another heart attack or of the death of cardiactissue. Aspirin may be effective at preventing certain types of cancer,particularly colorectal cancer.

Undesirable side effects of taking aspirin orally includegastrointestinal ulcers, stomach bleeding, and tinnitus, especially inhigher doses. In children and adolescents, aspirin is no longerindicated to control flu-like symptoms or the symptoms of chickenpox orother viral illnesses, because of the risk of Reye's syndrome.

Aspirin is part of a group of medications called nonsteroidalanti-inflammatory drugs (NSAIDs), but differs from most other NSAIDS inthe mechanism of action. Though aspirin, and others in its group calledthe salicylates, have similar effects (antipyretic, anti-inflammatory,analgesic) to the other NSAIDs and inhibit the same enzymecyclooxygenase, aspirin (but not the other salicylates) does so in anirreversible manner and, unlike others, affects more the COX-1 variantthan the COX-2 variant of the enzyme.

B. Sulindac and its Major Metabolites, Sulidac Sulfone and SulindacSulfide

Sulindac is a nonsteroidal, anti-inflammatory indene derivative with thefollowing chemical designation;(Z)-5-fluoro-2-methyl-1-((4-(methylsulfinyl)phenyl)methylene)-1H-indene-3-aceticacid (Physician's Desk Reference, 1999). Without being bound by theory,the sulfinyl moiety is converted in vivo by reversible reduction to asulfide metabolite and by irreversible oxidation to a sulfone metabolite(exisulind). See U.S. Pat. No. 6,258,845, which is incorporated hereinby reference. Sulindac, which also inhibits Ki-ras activation, ismetabolized to two different molecules, which differ in their ability toinhibit COX, yet both are able to exert chemopreventive effects via theinduction of apoptosis. Sulindac sulfone lacks COX-inhibitory activity,and most likely facilitates the induction of apoptosis in a mannerindependent of prostaglandin synthesis. Available evidence indicatesthat the sulfide derivative is at least one of the biologically activecompounds. Based on this, sulindac may be considered a prodrug.

Sulindac (Clinoril®) is available, for example, as 150 mg and 200 mgtablets. The most common dosage for adults is 150 to 200 mg twice a day,with a maximal daily dose of 400 mg. After oral administration, about90% of the drug is absorbed. Peak plasma levels are achieved in about 2hours in fasting patients and 3 to 4 hours when administered with food.The mean half-life of sulindac is 7.8 hours: the mean half-life of thesulfide metabolite is 16.4 hours. U.S. Pat. Nos. 3,647,858 and 3,654,349cover preparations of sulindac; both patents are incorporated byreference herein in their entireties.

Sulindac is indicated for the acute and long-term relief of signs andsymptoms of osteoarthritis, rheumatoid arthritis, ankylosingspondylitis, acute gout, and acute painful shoulder. The analgesic andanti-inflammatory effects exerted by sulindac (400 mg per day) arecomparable to those achieved by aspirin (4 g per day), ibuprofen (1200mg per day), indometacin (125 mg per day), and phenylbutazone (400 to600 mg per day). Side effects of sulindac include mild gastrointestinaleffects in nearly 20% of patients, with abdominal pain and nausea beingthe most frequent complaints. CNS side effects are seen in up to 10% ofpatients, with drowsiness, headache, and nervousness being those mostfrequently reported. Skin rash and pruritus occur in 5% of patients.Chronic treatment with sulindac can lead to serious gastrointestinaltoxicity such as bleeding, ulceration, and perforation.

The potential use of sulindac for chemoprevention of cancers, and inparticular colorectal polyps, has been well studied. For example, U.S.Pat. Nos. 5,814,625 and 5,843,929, which are both incorporated herein byreference, report potential chemopreventive uses of sulindac in humans.Sulindac has been shown to produce regression of adenomas in FamilialAdenomatous Polyposis (FAP) patients (Muscat et al., 1994), although atleast one study in sporadic adenomas has shown no such effect (Ladenheimet al., 1995). Sulindac and its sulfone metabolite exisulind have beentested and continue to be tested clinically for the prevention andtreatment of several cancer types.

C. Piroxicam

Piroxicam is a non-steroidal anti-inflammatory agent that is wellestablished in the treatment of rheumatoid arthritis and osteoarthritiswith the following chemical designation:4-hydroxy-2-methyl-N-2-pyridyl-2H-1,2-benzothiazine-3-carboxamide1,1-dioxide. Its usefulness also has been demonstrated in the treatmentof musculoskeletal disorders, dysmenorrhea, and postoperative pain. Itslong half-life enables it to be administered once daily. The drug hasbeen shown to be effective if administered rectally. Gastrointestinalcomplaints are the most frequently reported side effects.

Piroxicam has been shown to be an effective chemoprevention agent inanimal models (Pollard and Luckert, 1989; Reddy et al., 1987), althoughit demonstrated side effects in a recent IIb trial. A largemeta-analysis of the side effects of the NSAIDs also indicates thatpiroxicam has more side effects than other NSAIDs (Lanza et al., 1995).

The combination of DFMO and piroxicam has been shown to have asynergistic chemopreventive effect in the AOM-treated rat model of coloncarcinogenesis (Reddy et al., 1990), although DFMO exerted a greatersuppressive effect than piroxicam on Ki-ras mutation and tumorigenesiswhen each agent was administered separately (Reddy et al., 1990). In onestudy, administration of DFMO or piroxicam to AOM-treated rats reducedthe number of tumors harboring Ki-ras mutations from 90% to 36% and 25%,respectively (Singh et al., 1994). Both agents also reduced the amountof biochemically active p21 ras in existing tumors.

D. Celecoxib

Celecoxib is a non-steroidal anti-inflammatory agent that is wellestablished in the treatment of osteoarthritis, rheumatoid arthritis,acute pain, ankylosing spondylitis, and to reduce the number of colonand rectal polyps in patients with FAP with the following chemicaldesignation:4-[5-(4-Methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzenesulfonamide.Celecoxib is marketed under the brand names Celebrex, Celebra, andOnsenal by Pfizer. Celecoxib is a selective COX-2 inhibitor. Sideeffects of celecoxib include a 30% increase in rates of heart and bloodvessel disease. Additionally, the risks of gastrointestinal side effectsare greater than 80%.

E. Combinations of NSAIDs

Combinations of various NSAIDs may also be used in some embodiments. Byusing lower doses of two or more NSAIDs, it is possible, in someembodiments, to reduce the side effects or toxicities associated withhigher doses of individual NSAIDs. For example, in some embodiments,sulindac may be used together with celecoxib. Examples of NSAIDs thatmay be used in combination with one another include, but are not limitedto: ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen,oxaprozin, indomethacin, sulindac, etodolac, diclofenac, piroxicam,meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid,meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib,rofecoxib, valdecoxib, parecoxib, lumiracoxib, and etoricoxib.

IV. EFLORNITHINE/SULINDAC COMBINATION THERAPY

The compositions provided herein may be used, in some embodiments, toreduce the number of, inhibit the growth of, and/or prevent theoccurrence of cancer cells in patients. Target cancer cells includecancers of the lung, brain, prostate, kidney, liver, ovary, breast,skin, stomach, esophagus, head and neck, testicles, colon, cervix,lymphatic system and blood. In some embodiments, the compositions may beused to treat and/or prevent colon cancer, familial adenomatouspolyposis (FAP), pancreatic cancer, and/or neuroblastoma.

In some embodiments, the compositions provided herein may be used totreat patients exhibiting pre-cancerous symptoms and thereby prevent theonset of cancer. Target cells and tissues for such preventativetreatments include polyps and other precancerous lesions,premalignancies, preneoplastic, or other aberrant phenotype indicatingprobable progression to a cancerous state. For example, the compositionsprovided herein may be used to prevent adenomas with little additionaltoxicities. The Min (multiple intestinal neoplasia) mouse, which sharesa mutated APC/apc genotype with FAP, serves as a useful experimentalanimal model for human FAP patients (Lipkin, 1997). The Min mouse candevelop greater than 100 gastrointestinal adenomas/adenocarcinomasthroughout the gastrointestinal tract by 120 days of life leading to GIbleeding, obstruction and death. A combination therapy of DFMO andsulindac was shown to be effective in reducing adenomas in these mice.See U.S. Pat. No. 6,258,845, which is incorporated herein by referencein its entirety.

V. FIXED DOSE COMBINATIONS AND ROUTES OF ADMINISTRATION

In one aspect, the present invention provides compositions comprising afixed dose combination of a pharmaceutically effective amount ofeflornithine and a pharmaceutically effective amount of a nonsteroidalanti-inflammatory drug (NSAID) or a metabolite thereof. In someembodiments, the fixed dose combination is a pharmaceutically effectiveamount of eflornithine and a pharmaceutically effective amount ofsulindac.

In some embodiments, the eflornithine is eflornithine hydrochloridemonohydrate. In some embodiments, the eflornithine is eflornithinehydrochloride monohydrate racemate. In some embodiments, theeflornithine hydrochloride monohydrate is a racemic mixture of its twoenantiomers.

In some embodiments, the eflornithine is present in an amount of about10 to about 1000 mg. In some embodiments, the eflornithine is present inan amount of about 250 to about 500 mg. In some embodiments, theeflornithine is present in an amount of about 300 to about 450 mg. Insome embodiments, the eflornithine is present in an amount of about 350to about 400 mg. In some embodiments, the eflornithine is present in anamount of about 35 to about 60 weight percent. In some embodiments, theeflornithine is present in an amount of about 40 to about 55 weightpercent. In some embodiments, the eflornithine is present in an amountof about 50 to about 55 weight percent. In some embodiments, theeflornithine is present in an amount of about 52 to about 54 weightpercent. In some embodiments, the amount of eflornithine hydrochloridemonohydrate racemate is from 52 to 54 weight percent. In someembodiments, the eflornithine is present in an amount of about 375 mg.In some embodiments, the amount of eflornithine hydrochloridemonohydrate racemate is 375 mg.

In some embodiments, the sulindac is present in an amount from about 10to about 1500 mg. In some embodiments, the sulindac is present in anamount of about 50 to about 100 mg. In some embodiments, the sulindac ispresent in an amount of about 70 to about 80 mg. In some embodiments,the sulindac is present in an amount of about 75 mg. In someembodiments, the amount of sulindac is 75 mg. In some embodiments, thesulindac is present in an amount of about 5 to about 20 weight percent.In some embodiments, the sulindac is present in an amount of about 8 toabout 15 weight percent. In some embodiments, the sulindac is present inan amount of about 10 to about 12 weight percent. In some embodiments,the amount of sulindac is from 10 to 11 weight percent.

In some embodiments, the eflornithine is present in an amount of about375 mg and the sulindac is present in an amount of about 75 mg.

In some embodiments, the formulation further comprises an excipient. Insome embodiments, the excipient is starch, colloidal silicon dioxide, orsilicified microcrystalline cellulose. In some embodiments, theexcipient is colloidal silicon dioxide. In some embodiments, theformulation further comprises a second excipient. In some embodiments,the second excipient is silicified microcrystalline cellulose.

In some embodiments, the formulation further comprises a lubricant. Insome embodiments, the lubricant is magnesium stearate, calcium stearate,sodium stearate, glyceryl monostearate, aluminum stearate, polyethyleneglycol, boric acid or sodium benzoate. In some embodiments, thelubricant is magnesium stearate. In some embodiments, magnesium stearateis present in an amount of about 0.25 to about 2 weight percent. In someembodiments, the amount of magnesium stearate is from about 0.75 toabout 2 weight percent. In some embodiments, the amount of magnesiumstearate is from about 1 to about 1.5 weight percent. In someembodiments, the amount of magnesium stearate is about 1.1 weightpercent. In some embodiments, magnesium stearate is present in an amountof about 1.5 weight percent.

In some embodiments, the compositions are in the form of a capsule,tablet, mini tablets, granules, pellets, solution, gel, cream, foam orpatch. In some embodiments, the composition is in the form of a tablet,for example, a monolayer tablet.

In some embodiments, the weight of the tablet is from about 650 mg toabout 1,000 mg. In some embodiments, the weight of the tablet is fromabout 675 mg to about 725 mg. In some embodiments, the weight of thetablet is about 700 mg.

In some embodiments, the tablet further comprises a coating. In someembodiments, the coating is a modified release coating or an entericcoating. In some embodiments, the coating is a pH-responsive coating. Insome embodiments, the coating comprises cellulose acetate phthalate(CAP), cellulose acetate trimelletate (CAT), poly (vinyl acetate)phthalate (PVAP), hydroxypropylmethylcellulose phthalate (HP),poly(methacrylate ethylacrylate) (1:1) copolymer (MA-EA),poly(methacrylate methylmethacrylate) (1:1) copolymer (MA MMA),poly(methacrylate methylmethacrylate) (1:2) copolymer, orhydroxypropylmethylcellulose acetate succinate (HPMCAS). In someembodiments, the coating masks the taste of eflornithine. In someembodiments, the coating comprises hydroxypropyl methylcellulose,titanium dioxide, polyethylene glycol, and iron oxide yellow.

In some embodiments, the amount of coating is from about 1 to about 5weight percent. In some embodiments, the amount of coating is from about2 to about 4 weight percent. In some embodiments, the amount of coatingis about 3 weight percent. In some embodiments, the amount of coating isfrom about 5 mg to about 30 mg. In some embodiments, the amount ofcoating is from about 15 mg to about 25 mg. In some embodiments, theamount of coating is about 21 mg.

In some embodiments, the weight of the tablet comprising a coating isfrom about 675 mg to about 750 mg. In some embodiments, the weight ofthe tablet comprising a coating is from about 700 mg to about 725 mg. Insome embodiments, the weight of the tablet comprising a coating is about721 mg.

In one aspect, the present invention provides compositions comprising afixed dose combination of a pharmaceutically effective amount ofeflornithine and a pharmaceutically effective amount of sulindac. Insome embodiments, the compositions are in the form of a capsule, tablet,mini tablets, granules, pellets, solution, gel, cream, foam or patch. Insome embodiments, the compositions are solid and take the form of atablet, for example, a monolayer tablet. In some embodiments, the tabletis film coated.

In some aspects, the present disclosure provides oral fixed dosecombination formulations of eflornithine and an NSAID. In someembodiments, pharmaceutical compositions are provided that comprise apharmaceutically effective amount eflornithine and a pharmaceuticallyeffective amount of an NSAID. In some embodiments, the NSAID issulindac, aspirin, piroxicam or celecoxib. In some preferredembodiments, the NSAID is sulindac.

In some embodiments, the pharmaceutical compositions and formulations ofthe present invention are for enteral, such as oral, and also rectal orparenteral, with the compositions comprising the pharmacologicallyactive compounds either alone or together with pharmaceutical auxiliarysubstances (excipients). Pharmaceutical preparations for enteral orparenteral administration are, for example, in unit dose forms, such ascoated tablets, tablets, capsules or suppositories and also ampoules.These are prepared in a manner, which is known per se, for example usingconventional mixing, granulation, coating, solubilizing or lyophilizingprocesses. Thus, pharmaceutical preparations for oral use can beobtained by combining the active compounds with solid excipients, ifdesired granulating a mixture which has been obtained, and, if requiredor necessary, processing the mixture or granulate into tablets or coatedtablet cores after having added suitable auxiliary substances. In apreferred embodiment, a mixture of active ingredients and excipients areformulated into a tablet form. Appropriate coatings may be applied toincrease palatability or delay absorption. For example, a coating may beapplied to a tablet to mask the disagreeable taste of the activecompound, such as DFMO, or to sustain and/or to delay the release of theactive molecules to a certain area in the gastrointestinal tract.

The therapeutic compound can be orally administered, for example, withan inert diluent or an assimilable edible carrier. The therapeuticcompound and other ingredients may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet. For oral therapeutic administration, thetherapeutic compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, or wafers.

In certain embodiments, the tablets and/or capsules provided hereincomprise the active ingredients and powdered carriers, such as lactose,starch, cellulose derivatives, magnesium stearate, and stearic acid.Similar diluents can be used to make compressed tablets. In otherembodiments, tablets and capsules can be manufactured for immediate ormodified release. In some embodiments, the tablet and/or capsule ismanufactured as a sustained release product to provide for continuousrelease of medication over a period of hours. In some embodiments, thecompressed tablet is sugar-coated and/or film-coated to mask unpleasanttaste and/or protect the tablet from the atmosphere. In someembodiments, the tablet is enteric coated for selective disintegrationin the gastrointestinal tract.

In some embodiments, the tablet or capsule is able to disintegrate ordissolve to liberate multiparticulates comprising particles of differentpopulations of a first component and a second component, e.g. modifiedrelease coated multiparticles. In some of these embodiments, the tabletor capsule may disintegrate or dissolve in the mouth, stomach, smallintestine, terminal ileum, or colon. In some of these embodiments, thetablet or capsule may release the multiparticulates with modifiedrelease properties.

In some embodiments, the present invention provides a pharmaceuticaloral fixed dose combination in the form of a multilayer tablet. Amultilayer tablet has at least two layers (bilayer tablet) or can havethree, four, five or more layers. In some embodiments, each of thelayers contains not more than one of the active pharmaceuticalingredients (APIs). For example, in some embodiments, the tablet has twolayers, with one of the APIs in each of the two layers. In someembodiments, in addition to these two layers, the tablet containsfurther layers containing only carrier and which may function, e.g., asseparation layer(s) or outer coating layer(s). In some embodiments, ifmore than two layers are present, the components may be present in morethan one layer as long as they are not present together in the samelayer. In certain embodiments, a monolayer tablet is preferred but allinformation detailed below is equally applicable to multilayer tablets.

In some embodiments, the fixed dose combination may be formulated toprovide a mean steady state plasma concentration level of totaleflornithine and/or sulindac in the range of about 0.1 μM to about 1000μM and preferably in the range of about 1 μM to 100 μM and morepreferably in the range of about 1 μM to about 50 μM.

A. Pharmaceutically Acceptable Excipients

In some embodiments, the compositions further comprise apharmaceutically acceptable excipient. In some of these embodiments, thepharmaceutically acceptable excipient may include a pharmaceuticallyacceptable diluent, a pharmaceutically acceptable disintegrant, apharmaceutically acceptable binder, a pharmaceutically acceptablestabilizer, a pharmaceutically acceptable lubricant, a pharmaceuticallyacceptable pigment, or pharmaceutically acceptable glider. In a fixeddose combination formulation of the present invention, an activeingredient may be mixed at a weight ratio of 1:0.25 to 1:20 with apharmaceutically acceptable excipient.

Diluents that can be used in pharmaceutical formulations of the presentinvention include, but are not limited to, microcrystalline cellulose(“MCC”), silicified MCC (e.g. PROSOLV™ HD 90), microfine cellulose,lactose, starch, pregelatinized starch, sugar, mannitol, sorbitol,dextrates, dextrin, maltodextrin, dextrose, calcium carbonate, calciumsulfate, dibasic calcium phosphate dihydrate, tribasic calciumphosphate, magnesium carbonate, magnesium oxide, and any mixturesthereof. Preferably, the diluent is silicified MCC. The diluent may beused in an amount of from about 5 to about 95 weight percent based onthe total weight of the formulation, and preferably in an amount of fromabout 25 to about 40 percent weight, such as in an amount of from about30 to about 35 percent weight. In certain aspects, the diluent can be asoluble diluent. When the diluent is used, its ratio to the activeingredient in each discrete layer is very important. The term “solublediluents” refers to a diluent which is dissolved in water, like lactose,Ludipress (BASF, a mixture of lactose, crospovidone and povidone(93:3.5:3.5, w/w (%))), mannitol and sorbitol.

Disintegrants are used to promote swelling and disintegration of thetablet after exposure to fluids in the oral cavity and/orgastrointestinal tract. Examples of disintegrants useful in the fixeddose combination formulation of the present invention includecrospovidone, sodium starch glycolate, croscarmellose sodium,low-substituted hydroxypropylcellulose, starch, alginic acid or sodiumsalt thereof, and a mixture thereof. Other disintegrants that can beused in pharmaceutical formulations of the present invention include,but are not limited to, methylcelluloses, microcrystalline celluloses,carboxymethyl cellulose calcium, carboxymethyl cellulose sodium (e.g.AC-DI-SOL™, PRIMELLOSE™), povidones, guar gum, magnesium aluminumsilicate, colloidal silicon dioxide (e.g. AEROSIL™, CARBOSIL™),polacrilin potassium, starch, pregelatinized starch, sodium starchglycolate (e.g. EXPLOTAB™), sodium alginate, and any mixtures thereof.Preferably, the disintegrant is colloidal silicon dioxide. Thedisintegrant may be used in an amount of about 0.1 to about 30 weightpercent based on the total weight of the formulation, and preferably inan amount of about 0.2 to about 5 weight percent.

Compositions of the present invention may comprise lubricants. Stickingcan occur when granules attach themselves to the faces of tablet presspunches. Lubricants are used to promote flowability of powders, and toreduce friction between the tablet punch faces and the tablet punchesand between the tablet surface and the die wall. For example, lubricantsinclude magnesium stearate, calcium stearate, zinc stearate, stearicacid, sodium stearyl fumarate, polyethylene glycol, sodium iaurylsulphate magnesium lauryl sulphate, and sodium benzoate. Preferably, thelubricant is magnesium stearate. In the present invention, lubricantspreferably comprise 0.25 weight percent to 2 weight percent of the soliddosage form, and preferably in an amount of about 1.5 weight percent. Inan exemplary formulation, the lubricant is magnesium stearate present inan amount of about 1.5 weight percent to prevent sticking.

Binders can be used in the pharmaceutical compositions of the presentinvention to help hold tablets together after compression. Examples ofbinders useful for the present invention are acacia, guar gum, alginicacid, carbomers (e.g. Carbopol™ products), dextrin, maltodextrin,methylcelluloses, ethylcelluloses, hydroxyethyl celluloses,hydroxypropyl celluloses (e.g. KLUCEL™), hydroxypropyl methylcelluloses(e.g. METHOCEL™), carboxymethylcellulose sodiums, liquid glucose,magnesium aluminum silicate, polymethacrylates, polyvinylpyrrolidones(e.g., povidone K-90 D, KOLLIDON™), copovidone (PLASDONE™), gelatin,starches, and any mixtures thereof. Preferably, the binder is starch. Inthe present invention, binders preferably comprise about 1 to about 15weight percent of the solid dosage form. In other embodiments, the soliddosage form does not comprise a binder.

In certain embodiments, the stabilizer usable in the fixed dosecombination formulation of the present invention may be an anti-oxidant.The use of an antioxidant enhances stability of the active ingredientsagainst the undesirable reaction with other pharmaceutically acceptableadditives and against modification by heat or moisture with time. Forexample, the anti-oxidant is ascorbic acid and its esters, butylatedhydroxytoluene (BHT), butylated hydroxyanisole (BHA), α-tocopherol,cystein, citric acid, propyl gallate, sodium bisulfate, sodiumpyrosulfite, ethylene diamine tetracetic acid (EDTA), and any mixturesthereof.

B. Tablet Manufacture Processes

A further aspect of the present invention is providing processes for themanufacturing tablets disclosed herein, including those comprisingeflornithine and sulindac. In some embodiments, active agents areprepared by sifting at least one active agent and one or more excipientsthrough a desired mesh size sieve and then mixing, using a rapid mixergranulator, planetary mixer, mass mixer, ribbon mixer, fluid bedprocessor, or any other suitable device. The blend can be granulated,such as by adding a solution or suspension with or without a binder,whether alcoholic or hydro-alcoholic or aqueous, in a low or high shearmixer, fluidized bed granulator and the like, or by dry granulation. Thegranules can be dried using a tray dryer, fluid bed dryer, rotary conevacuum dryer, and the like. The granules can be sized using anoscillating granulator or comminuting mill or any other conventionalequipment equipped with a suitable screen. Alternatively, granules canbe prepared by extrusion and spheronization, or roller compaction. Also,the manufacture of granules containing active agents can include mixingwith directly compressible excipients or roller compaction.

In other embodiments of the invention, small tablets (mini-tablets) canbe made by compressing granules, using dies and punches of various sizesand shapes, as desired. Optionally, a coating can be applied to thetablets, if desired, by techniques known to one skilled in the art suchas spray coating, dip coating, fluidized bed coating and the like. Incertain embodiments of the present invention, suitable solvent systemssuch as alcoholic, hydroalcoholic, aqueous, or organic may be used tofacilitate processing.

1. Granulation

Granulation is a process in which powder particles are made to adhere toeach other, resulting in larger, multi-particle entities or granules. Inembodiments of the invention, granules obtained by a dry or wettechnique can be blended with one or more lubricants and/oranti-adherants and then filled into single capsule or into differentcapsules of different sizes, such that a smaller capsule can be filledinto another larger capsule.

In certain embodiment, dry granulation by compaction is used for theproduction of the solid dosage composition. In dry granulation, thepowder blend is compacted by applying a force onto the powder, which isgeneral causes a considerable size enlargement. In some aspects,slugging is used in the dry granulation process in which a tablet pressis used for the compaction process. In other aspects, a roller compactoris used for dry granulation including a feeding system, compaction unitand size reduction unit. In this method, the powder is compacted betweentwo rolls by applying a force, which is the most important parameter inthe dry granulation process. The applied force is expressed in kN/cm,being the force per cm roll width. Occasionally the press force is alsoindicated in bar. This, however, merely represents the pressure withinthe hydraulic system, and is in fact not an appropriate measuring unitfor the force applied onto the powder. At a given force, depending onthe amount of powder conveyed to the rolls, the powder will be compactedto a predefined ribbon thickness.

In other embodiments, wet granulation is used for the production of thesolid dosage composition. Wet granulation of powders improves flow andcompactability of the compression mix. In wet granulation, granules areformed by the addition of a granulation liquid onto a powder bed, whichis under the influence of an impeller (in a high-shear granulator),screws (in a twin screw granulator) or air (in a fluidized bedgranulator). The agitation resulting in the system along with thewetting of the components within the formulation results in theaggregation of the primary powder particles to produce wet granules. Thegranulation liquid (fluid) contains a solvent, which must be volatile sothat it can be removed by drying, and be non-toxic. Typical liquidsinclude water, ethanol and isopropanol either alone or in combination.The liquid solution can be either aqueous based or solvent-based.Aqueous solutions have the advantage of being safer to deal with thanorganic solvents.

Tablets may also be formed by tumbling melt granulation (TMG)essentially as described in Maejima et al, 1997; which is incorporatedherein by reference. Tumbling melt granulation can be used for preparingthe melt granulation. It can be done in a tumbling mixer. The molten lowmelting point compound is sprayed on the crystalline saccharide andpowdered saccharide in the blender and are mixed until granules form. Inthis case, the low melting ingredient is the binder and the crystallinesaccharide is the seed. An alternative method is to combine the unmeltedlow melting point ingredient, crystalline sugar (e.g. sucrose ormaltose), and water-soluble ingredient in the powder form (e.g.,mannitol or lactose) in the tumbling mixer and mix while heating to themelting point of the low melting point binder or higher. The seed shouldbe crystalline or granular water soluble ingredient (saccharide), e.g.,granular mannitol, crystalline maltose, crystalline sucrose, or anyother sugar. An example of tumbling mixers is the twin-shell blender(V-blender), or any other shape of tumbling mixers. Heating can beachieved by circulating heated air through the chamber of the granulatorand by heating the bottom surface of the chamber. As the seed materialand the powdered tablet constituents circulate in the heated chamber,the low-melting point compound melts and adheres to the seeds. Theunmelted, powdered material adheres to the seed-bound, moltenlow-melting point material. The spherical beads, which are formed bythis process are then cooled and screen sifted to remove nonadheredpowder.

Spray congealing or prilling can also be used to form the tabletcompositions of the invention. Spray congealing includes atomizingmolten droplets of compositions which include a low melting pointcompound onto a surface or, preferably, other tablet constituents.Equipment that can be used for spray congealing includes spray driers(e.g., Nero spray drier) and a fluid bed coater/granulation with topspray (e.g., Glatt fluid bed coater/granulator). In preferredembodiments, a fast-dissolve granulation is formed wherein, preferably awater soluble excipient, more preferably a saccharide, is suspended in amolten low melting point ingredient and spray congealed. After spraycongealing, the resulting composition is allowed to cool and congeal.Following congealing of the mixture, it is screened or sieved and mixedwith remaining tablet constituents. Spray congealing processes whereinfast-dissolve granulations comprising any combination of low meltingpoint compound and other tablet constituents are melted and spraycongealed onto other tablet constituents are within the scope of thepresent invention. Spray congealing processes wherein all tabletconstituents, including the low-melting point compound, are mixed, thelow melting point compound is melted and the mixture is spray congealedonto a surface are also within the scope of the invention.

2. Blending

In certain embodiments, the mixture is blended after granulation.Blending in solid dose manufacturing is to achieve blend uniformity andto distribute the lubricant. In certain aspects, the blend step(s) aredesigned to achieve homogeneity of all components prior to the finalblend of the lubricant. However, blending powders is a challenge due toparticle size, moisture content, structure, bulk density and flowcharacteristics. The key to a successful formula is the order ofaddition. Typically the component and pharmaceutically acceptableadditives are dispatched to a suitable vessel such as a diffusionblender or diffusion mixer. An example of tumbling mixers is thetwin-shell blender (V-blender), or any other shape of tumbling mixers.

3. Compression

Once tablet compositions are prepared, they may be formed into variousshapes. In preferred embodiments, the tablet compositions are pressedinto a shape. This process may comprise placing the tablet compositioninto a form and applying pressure to the composition so as to cause thecomposition to assume the shape of the surface of the form with whichthe composition is in contact. Compression into a tablet form can beaccomplished by a tablet press. A tablet press includes a lower punchthat fits into a die from the bottom and an upper punch having acorresponding shape and dimension which enters the die cavity from thetop after the tableting material falls into the die cavity. The tabletis formed by pressure applied on the lower and upper punches. Thetablets of the invention generally have a hardness of about 20 kP orless; preferably the tablets have a hardness of about 15 kP or less.Typical compression pressures are about 5 kN to about 40 kN and willvary based on the desired size and hardness of the tablet. In someaspects, the compression pressure is about 25 kN to about 35 kN. Inparticular aspects, the compression pressure is less than or about 37kN, such as less than about 30 kN, such as less than about 25 kN.Hydraulic presses such as a Carver Press or rotary tablet presses suchas the Stokes Versa Press are suitable means by which to compress thetablet compositions of the invention. Exemplary compression forceparameters are shown in Table 3.

In certain embodiments, the lubricated blend can be compressed using asuitable device, such as a rotary machine to form slugs, which arepassed through a mill or fluid energy mill or ball mill or colloid millor roller mill or hammer mill and the like, equipped with a suitablescreen to obtain the milled slugs of actives.

A pre-compression step can be used such as to prevent capping of thetablet. Capping refers to the split or fracture of the cap or top of atablet from the body of the tablet. Capping can be caused bynon-compressible fine particles that migrate when the air is pushed outduring compression. For example, the pre-compression can be at about 5,10 or 15 percent of the main compression force. In preferredembodiments, the tablet is pre-compressed into the form at a pressure,which will not exceed about 10 kN, preferably less than 5 kN. Forexample, pressing the tablets at less than 1, 1.5, 2, 2.1, 2.2, 2.5, 3,3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 kN is within the scope of theinvention. In particular aspects, the pre-compression force is about 2.5kN to about 3.5 kN. Exemplary pre-compression force parameters are shownin Table 3.

4. Film Coating

The composition or solid dosage form according to the invention may alsobe coated with a film coating, an enteric coating, a modified releasecoating, a protective coating, or an anti-adhesive coating.

The composition of the invention may be enteric coated. By entericcoated or coating is meant a pharmaceutically acceptable coatingpreventing the release of the active agent in the stomach and allowingthe release in the upper part of the intestinal tract. In otherembodiments, the enteric coating is applied to delay the release of theactive agent to the terminal ileum or to the colon. The enteric coatingmay be added as an overcoat upon the modified release coating. Theenteric coating polymers can be used either alone or in combination inthe enteric coating formulation. Enteric coatings can be designed as asingle layer or as multilayer coating embodiments. The preferred entericcoating for the composition of the invention comprises a film-formingagent selected from cellulose acetate phthalate; cellulose acetatetrimellitate; methacrylic acid copolymers, copolymers derived frommethylacrylic acid and esters thereof, containing at least 40%methylacrylic acid; hydroxypropyl methylcellulose phthalate;hydroxypropylmethylcellulose acetate succinate orPolyvinylacetatephthalate. Examples of polymers suitable for entericcoating include, for example, cellulose acetate phthalate (CAP),cellulose acetate trimelletate (CAT), poly (vinyl acetate) phthalate(PVAP), hydroxypropylmethylcellulose phthalate (HP), poly(methacrylateethylacrylate) (1:1) copolymer (MA-EA), poly(methacrylatemethylmethacrylate) (1:1) copolymer (MA MMA), poly(methacrylatemethylmethacrylate) (1:2) copolymer, EUDRAGIT™ L 30D (MA-EA, 1:1),EUDRAGIT™ 100 55 (MA-EA, 3:1), hydroxypropylmethylcellulose acetatesuccinate (HPMCAS), SURETERIC (PVAP), AQUATERIC™ (CAP), shellac orAQOAT™ (HPMCAS). Targeted colonic delivery systems which may be usedwith the present invention are known and employ materials such ashydroxypropylcellulose, microcrystalline cellulose (MCE, AVICEL™ fromFMC Corp.), poly(ethylene-vinyl acetate) (60:40) copolymer (EVAC fromAldrich Chemical Co.), 2-hydroxyethylmethacrylate (HEMA), MMA,terpolymers of HEMA:MMA:MA synthesized in the presence ofN,N′-bis(methacryloyloxyethyloxycarbonylamino)-azobenzene, azopolymers,enteric coated timed release system (TIME CLOCK® from PharmaceuticalProfiles, Ltd., UK) and calcium pectinate and the osmotic minipumpsystem (ALZA corp.).

In some embodiments, the film coating comprises a polymer such ashydroxypropylcellulose (HPC), ethylcellulose (EC),hydroxypropylmethylcellulose (HMPC), hydroxyethylcellulose (HEC), sodiumcarboxymethylcellulose (CMC), poly(vinyl pyrrolidone) (PVP),poly(ethylene glycol) (PEG), dimethylaminoethyl methacrylate-methacrylicacid ester copolymer, or ethylacrylate-methylmethacrylate copolymer(EA-MMA).

In some embodiments, the composition has a modified release coating. Themodified release coating may be a pH-responsive coating which whenexposed to a certain pH will deliver the active agent(s) (e.g., to thecolorectal tract). In some embodiments, the pH-responsive coating is apH-responsive polymer that will dissolve when exposed to a pH greaterthan or equal to about 6; although, the pH-responsive polymer maydissolve at a pH greater than or equal to about 5. The pH-responsivepolymer may be, for example, a polymeric compound such as EUDRAGIT™ RSand EUDRAGIT™ RL. The EUDRAGIT™ products form latex dispersions of about30D by weight. EUDRAGIT™ RS 30D is designed for slow release since it isnot very water permeable as a coating and EUDRAGIT™ RS 30D is designedfor rapid release since it is relatively water permeable as a coating.These two polymers are generally used in combination. As contemplatedherein, the permissible ratios of EUDRAGIT™ RS 30D/EUDRAGIT™ RL 30D isabout 10:0 to about 8:2. Ethylcellulose or S100 or other equivalentpolymers designed for enteric or colorectal release can also be used inplace of the EUDRAGIT™ RS/EUDRAGIT™ RL combination above.

Optionally, the method comprises the step of film coating the tablet.Film coating can be accomplished using any suitable means. Suitable filmcoatings are known and commercially available or can be made accordingto known methods. Typically the film coating material is a polymericfilm coating material comprising materials such as polyethylene glycol,talc and colorant. Suitable coating materials are methylcellulose,hydroxypropylmethylcellulose, hydroxypropylcellulose, acrylic polymers,ethylcellulose, cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, polyvinylalcohol,sodium carboxymethylcellulose, cellulose acetate, cellulose acetatephthalate, gelatin, methacrylic acid copolymer, polyethylene glycol,shellac, sucrose, titanium dioxide, camauba wax, microcrystalline wax,and zein. In some aspects, the film coating is hydroxypropylmethylcellulose, titanium dioxide, polyethylene glycol, and iron oxideyellow. For example, the film coating is OPADRY® Yellow (Colorcon).Typically, a film coating material is applied in such an amount as toprovide a film coating that ranges of from 1% to 6% by weight of thefilm-coated tablet, such as from 2% to 4%, such as about 3%.Plasticizers and other ingredients may be added in the coating material.The same or different active substance may also be added in the coatingmaterial.

In some embodiments, the coating of the tablet can improve palatabilitysuch as to mask the disagreeable taste of the active ingredient(s) suchas DFMO. For example, the tablet coating composition can include acellulose polymer, a plasticizer, a sweetener, or a powdered flavorcomposition, the powdered flavor composition including a flavorantassociated with a solid carrier.

C. Administration Schedules and Protocols

In some embodiments, the agent(s) may be administered on a routineschedule. As used herein, a routine schedule refers to a predetermineddesignated period of time. The routine schedule may encompass periods oftime which are identical or which differ in length, as long as theschedule is predetermined. For instance, the routine schedule mayinvolve administration twice a day, every day, every two days, everythree days, every four days, every five days, every six days, a weeklybasis, a monthly basis or any set number of days or weeks there-between.Alternatively, the predetermined routine schedule may involveadministration on a twice daily basis for the first week, followed by adaily basis for several months, etc. In other embodiments, the inventionprovides that the agent(s) may be taken orally and that the timing ofwhich is or is not dependent upon food intake. Thus, for example, theagent can be taken every morning and/or every evening, regardless ofwhen the subject has eaten or will eat.

VI. DIAGNOSIS AND TREATMENT OF PATIENTS

In some embodiments, the treatment methods may be supplemented withdiagnostic methods to improve the efficacy and/or minimize the toxicityof the anti-cancer therapies comprising administration of thecompositions provided herein. Such methods are described, for example,in U.S. Pat. Nos. 8,329,636 and 9,121,852, U.S. Patent PublicationsUS2013/0217743 and US2015/0301060, and PCT Patent PublicationsWO2014/070767 and WO2015/195120, which are all incorporated herein byreference.

In some embodiments, compositions and formulations of the presentdisclosure may be administered to a subject with a genotype at position+316 of at least one allele of the ODC1 gene promoter is G. In someembodiments, the genotype at position +316 of both alleles of thepatient's ODC1 gene promoters may be GG. In some embodiments, thegenotype at position +316 of both alleles of the patient's ODC1 genepromoters may be GA. A statistically significant interaction wasdetected for ODC1 genotype and treatment in a full model for adenomarecurrence, such that the pattern of adenoma recurrence among placebopatients was: GG 50%, GA 35%, AA 29% versus eflornithine/sulindacpatients: GG 11%, GA 14%, AA 57%. The adenoma-inhibitory effect ofeflornithine and sulindac was greater among those with the major Ghomozygous ODC1 genotype, in contrast to prior reports showing decreasedrisk of recurrent adenoma among CRA patients receiving aspirin carryingat least one A allele (Martinez et al., 2003; Barry et al., 2006; Hubneret al., 2008). These results demonstrate that ODC1 A allele carriers atposition +316 differ in response to prolonged exposure with eflornithineand sulindac compared to GG genotype patients, with A allele carriersexperiencing less benefit in terms of adenoma recurrence, and potentialfor elevated risk of developing ototoxicity, especially among the AAhomozygotes.

In some embodiments, the invention provides methods for the preventativeor curative treatment of colorectal carcinoma in a patient comprising:(a) obtaining results from a test that determines the patient's genotypeat position +316 of at least one ODC1 promoter gene allele; and (b) ifthe results indicate that the patient's genotype at position +316 of atleast one allele of the ODC1 promoter gene is G, then administering tothe patient a composition provided herein. In some embodiments, theinvention provides methods for the treatment of colorectal carcinomarisk factors in a patient comprising: (a) obtaining results from a testthat determines the patient's genotype at position +316 of at least oneODC1 promoter gene allele; and (b) if the results indicate that thepatient's genotype at position +316 of at least one allele of the ODC1promoter gene is G, then administering to the patient a compositionprovided herein, wherein the methods prevent the formation of newaberrant crypt foci, new adenomatous polyps or new adenomas withdysplasia in the patient. See U.S. Pat. No. 8,329,636, which isincorporated herein by reference.

In some embodiments, the invention provides methods for the preventativeor curative treatment of familial adenomatous polyposis (FAP) orneuroblastoma in a patient comprising: (a) obtaining results from a testthat determines the patient's genotype at position +316 of at least oneODC1 promoter gene allele; and (b) if the results indicate that thepatient's genotype at position +316 of at least one allele of the ODC1promoter gene is G, then administering to the patient a compositionprovided herein. In some embodiments, the invention provides methods forthe treatment of familial adenomatous polyposis or neuroblastoma riskfactors in a patient comprising: (a) obtaining results from a test thatdetermines the patient's genotype at position +316 of at least one ODC1promoter gene allele; and (b) if the results indicate that the patient'sgenotype at position +316 of at least one allele of the ODC1 promotergene is G, then administering to the patient a composition providedherein, wherein the methods prevent the formation of new aberrant cryptfoci, new adenomatous polyps or new adenomas with dysplasia in thepatient. See U.S. Pat. No. 9,121,852, which is incorporated herein byreference.

In some embodiments, the invention provides methods for treatingpatients with carcinoma comprising administering to the patients acomposition provided herein, wherein the patients have been determinedto have a dietary polyamine intake, and/or tissue polyamine level,and/or tissue polyamine flux that is not high. In some of theseembodiments, the dietary polyamine intake that is not high is 300 μMpolyamine per day or lower. In some of these embodiments, the carcinomais colorectal cancer. See U.S. Patent Publication US2013/0217743, whichis incorporated herein by reference.

In some embodiments, the invention provides methods for the preventativeor curative treatment of cancer in a patient comprising: (a) obtainingresults from a test that determines an expression level of a let-7non-coding RNA, a HMGA2 protein, and/or a LIN28 protein in a cancer cellfrom the patient; and (b) if the results indicate that the patient'scancer exhibits a reduced let-7 non-coding RNA expression level ascompared to a reference let-7 non-coding RNA expression level, anelevated HMGA2 protein expression level as compared a reference HMGA2protein expression level, and/or an elevated LIN28 protein expressionlevel as compared to a reference LIN28 protein expression level, thenadministering to the patient a composition provided herein. In some ofthese embodiments, the reference level is a level observed in anon-diseased subject or a level observed in a non-cancerous cell fromthe patient. In some of these embodiments, “obtaining” comprisesproviding a sample of the cancer from the patient and assessing anexpression level of a let-7 non-coding RNA, an HMGA2 protein, or a LIN28protein in a cancer cell from the sample. In some of these embodiments,“assessing an expression level of a let-7 non-coding RNA” comprisesquantitative PCR or Northern blotting. In some of these embodiments,“assessing an expression level of a HMGA2 protein or a LIN28 protein”comprises immunohistochemistry or ELISA. In some of these embodiments,the sample is blood or tissue, such as tumor tissue. In some of theseembodiments, the patient is a human. In some of these embodiments, thecancer is colorectal cancer, neuroblastoma, breast cancer, pancreaticcancer, brain cancer, lung cancer, stomach cancer, a blood cancer, skincancer, testicular cancer, prostate cancer, ovarian cancer, livercancer, esophageal cancer, cervical cancer, head and neck cancer,non-melanoma skin cancer, or glioblastoma. In some of these embodiments,the methods further comprise (c) obtaining results from a test thatdetermines the expression of a let-7 non-coding RNA in a second cancercell from said patient at a second time point following theadministration of at least one dose of the ODC inhibitor. In some ofthese embodiments, the methods further comprise increasing the amount ofthe ODC inhibitor administered to the patient if no or a small increasein let-7 non-coding RNA is observed. In some of these embodiments, themethods further comprise obtaining results from a test that determinesthe expression of a HMGA2 protein or a LIN28 protein in a second cancercell from said patient at a second time point following theadministration of at least one dose of the ODC inhibitor. In some ofthese embodiments, the methods further comprise increasing the amount ofthe ODC inhibitor administered to the patient if no or a small decreasein HMGA2 protein or LIN28 protein is observed. In some of theseembodiments, the methods further comprise (i) obtaining results from atest that determines the patient's genotype at position +316 of at leastone allele of the ODC1 gene promoter; and (ii) if the results indicatethat the patient's genotype at position +316 of at least one allele ofthe ODC1 gene promoter is G, then administering to the patient acomposition provided herein. In some embodiments, the methods comprisediagnosing a cancer or precancerous condition in a patient comprisingobtaining a sample from the patient and (b) determining an expressionlevel of at least two markers selected from the group consisting of alet-7 non-coding RNA, a LIN28 protein, and a HMGA2 protein in thesample, wherein if the expression level of the let-7 non-coding RNA isdecreased or the LIN28 protein or HMGA2 protein is increased in thesample relative to a reference level, then the patient is diagnosed ashaving cancer or a precancerous condition. In some embodiments, thefixed dose combination of the present invention is administered to apatient with a low cell or tissue let-7 level. In other aspects, thepresent compositions are administered to a patient with a high cell ortissue HMGA2 level. In other aspects, the compositions of the presentinventions are administered to a patient with a high cell or tissueLIN28 level. See U.S. Patent Publication US2015/0301060, which isincorporated herein by reference.

In some embodiments, there are provided methods for the preventative orcurative treatment of carcinoma in a patient comprising: (a) obtainingresults from a test that determines the patient's genotype at position+263 of at least one ODC1 allele; and (b) if the results indicate thatthe patient's genotype at position +263 of at least one allele of theODC1 gene is T, then administering to the patient a composition providedherein. In some of these embodiments, the test may determine thenucleotide base at position +263 of one allele of the ODC1 gene in thepatient. In some embodiments, the test may determine the nucleotidebases at position +263 of both alleles of the ODC1 gene in the patient.In some embodiments, the results may indicate that the patient'sgenotype at position +263 of both alleles of the ODC1 gene is TT. Insome embodiments, the results may indicate that the patient's genotypeat position +263 of both alleles of the ODC1 gene is TG. In some ofthese embodiments, the method may further comprise obtaining resultsfrom a test that determines the patient's genotype at position +316 ofat least one ODC1 allele and only administering to the patient of thecomposition provided herein if the results indicate that the patient'sgenotype at position +316 of at least one allele of the ODC1 gene is G.In another aspect, there are provided methods for the treatment ofcolorectal carcinoma risk factors in a patient comprising: (a) obtainingresults from a test that determines the patient's genotype at position+263 of at least one ODC1 allele; and (b) if the results indicate thatthe patient's genotype at position +263 of at least one allele of theODC1 gene is T, then administering to the patient a composition providedherein, wherein the method prevents the formation of new aberrant cryptfoci, new adenomatous polyps or new adenomas with dysplasia in thepatient. In another aspect, there is provided methods for preventing thedevelopment or recurrence of a carcinoma in a patient at risk thereforcomprising: (a) obtaining results from a test that determines thepatient's genotype at position +263 of at least one ODC1 allele; and (b)if the results indicate that the patient's genotype at position +263 ofat least one allele of the ODC1 gene is T, then administering to thepatient a composition provided herein. See PCT Patent PublicationWO2015/195120, which is incorporated herein by reference.

In variations on any of the above embodiments, the carcinoma may becolorectal cancer, neuroblastoma, breast cancer, pancreatic cancer,brain cancer, lung cancer, stomach cancer, a blood cancer, skin cancer,testicular cancer, prostate cancer, ovarian cancer, liver cancer,esophageal cancer, cervical cancer, head and neck cancer, non-melanomaskin cancer, or glioblastoma. In some embodiments, the carcinoma may becolorectal cancer. In some embodiments, the colorectal cancer may bestage I. In some embodiments, the colorectal cancer may be stage II. Insome embodiments, the colorectal cancer may be stage III. In someembodiments, the colorectal cancer may be stage IV. In variations on anyof the above embodiments, the methods may prevent the formation of newadvanced colorectal neoplasms within the patient. In some embodiments,the method may prevent the formation of new right-sided advancedcolorectal neoplasms. In some embodiments, the method may prevent theformation of new left-sided advanced colorectal neoplasms.

In variations on any of the above embodiments, the patient may have beenidentified as having one or more adenomatous polyps in the colon, rectumor appendix. In some embodiments, the patient may have been identifiedas having one or more advanced colorectal neoplasms. In someembodiments, the patient may have been identified as having one or moreleft-side advanced colorectal neoplasms. In some embodiments, thepatient may have been identified as having one or more right-sidedadvanced colorectal neoplasms. In some embodiments, the patient may havebeen diagnosed with familial adenomatous polyposis. In some embodiments,the patient may have been diagnosed with Lynch syndrome. In someembodiments, the patient may have been diagnosed with familialcolorectal cancer type X. In some embodiments, the patient may satisfythe Amsterdam Criteria or the Amsterdam Criteria II. In someembodiments, the patient may have a history of resection of one or morecolorectal adenomas. In some embodiments, the patient may have anintraepithelial neoplasia or a precancerous lesion associated ODChyperactivity. In some embodiments, the patient may have anintraepithelial neoplasia or a precancerous lesion and elevated cellularpolyamine levels.

In variations on any of the above embodiments, the patient is human

VII. DEFINITIONS

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

As used herein, the term “bioavailability” denotes the degree means towhich a drug or other substance becomes available to the target tissueafter administration. In the present context, the term “suitablebioavailability” is intended to mean that administration of acomposition according to the invention will result in a bioavailabilitythat is improved compared to the bioavailability obtained afteradministration of the active substance(s) in a plain tablet; or thebioavailability is at least the same or improved compared to thebioavailability obtained after administration of a commerciallyavailable product containing the same active substance(s) in the sameamounts. In particular, it is desired to obtain quicker and largerand/or more complete uptake of the active compound, and thereby providefor a reduction of the administered dosages or for a reduction in thenumber of daily administrations.

The terms “compositions,” “pharmaceutical compositions,” “formulations,”and “preparations” are used synonymously and interchangeably herein.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “derivative thereof” refers to any chemically modifiedpolysaccharide, wherein at least one of the monomeric saccharide unitsis modified by substitution of atoms or molecular groups or bonds. Inone embodiment, a derivative thereof is a salt thereof. Salts are, forexample, salts with suitable mineral acids, such as hydrohalic acids,sulfuric acid or phosphoric acid, for example hydrochlorides,hydrobromides, sulfates, hydrogen sulfates or phosphates, salts withsuitable carboxylic acids, such as optionally hydroxylated loweralkanoic acids, for example acetic acid, glycolic acid, propionic acid,lactic acid or pivalic acid, optionally hydroxylated and/oroxo-substituted lower alkanedicarboxylic acids, for example oxalic acid,succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid,pyruvic acid, malic acid, ascorbic acid, and also with aromatic,heteroaromatic or araliphatic carboxylic acids, such as benzoic acid,nicotinic acid or mandelic acid, and salts with suitable aliphatic oraromatic sulfonic acids or N-substituted sulfamic acids, for examplemethanesulfonates, benzenesulfonates, p-toluenesulfonates orN-cyclohexylsulfamates (cyclamates).

The term “disintegration” as used herein refers to a process where thepharmaceutical oral fixed dose combination, typically by means of afluid, falls apart into separate particles and is dispersed.Disintegration is achieved when the solid oral dosage form is in a statein which any residue of the solid oral dosage form, except fragments ofinsoluble coating or capsule shell, if present, remaining on the screenof the test apparatus is a soft mass having no palpably firm core inaccordance with USP<701>. The fluid for determining the disintegrationproperty is water, such as tap water or deionized water. Thedisintegration time is measured by standard methods known to the personskilled in the art, see the harmonized procedure set forth in thepharmacopeias USP <701> and EP 2.9.1 and JP.

The term “dissolution” as used herein refers to a process by which asolid substance, here the active ingredients, is dispersed in molecularform in a medium. The dissolution rate of the active ingredients of thepharmaceutical oral fixed dose combination of the invention is definedby the amount of drug substance that goes in solution per unit timeunder standardized conditions of liquid/solid interface, temperature andsolvent composition. The dissolution rate is measured by standardmethods known to the person skilled in the art, see the harmonizedprocedure set forth in the pharmacopeias USP <711> and EP 2.9.3 and JP.For the purposes of this invention, the test is for measuring thedissolution of the individual active ingredients is performed followingpharmacopoeia USP <711> at the pH as set forth herein for the differentembodiments. In particular, the test is performed using a paddlestirring element at 75 rpm (rotations per minute). The dissolutionmedium is preferably a buffer, typically a phosphate buffer (e.g., at pH7.2). The molarity of the buffer is preferably 0.1 M.

An “active ingredient” (AI) (also referred to as an active compound,active substance, active agent, pharmaceutical agent, agent,biologically active molecule, or a therapeutic compound) is theingredient in a pharmaceutical drug or a pesticide that is biologicallyactive. The similar terms active pharmaceutical ingredient (API) andbulk active are also used in medicine, and the term active substance maybe used for pesticide formulations.

A “pharmaceutical drug” (also referred to as a pharmaceutical,pharmaceutical preparation, pharmaceutical composition, pharmaceuticalformulation, pharmaceutical product, medicinal product, medicine,medication, medicament, or simply a drug) is a drug used to diagnose,cure, treat, or prevent disease. An active ingredient (AI) (definedabove) is the ingredient in a pharmaceutical drug or a pesticide that isbiologically active. The similar terms active pharmaceutical ingredient(API) and bulk active are also used in medicine, and the term activesubstance may be used for pesticide formulations. Some medications andpesticide products may contain more than one active ingredient. Incontrast with the active ingredients, the inactive ingredients areusually called excipients in pharmaceutical contexts.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult. “Effective amount,” “therapeutically effective amount” or“pharmaceutically effective amount” when used in the context of treatinga patient or subject with a compound means that the amount of thecompound which, when administered to a subject or patient for treatingor preventing a disease, is an amount sufficient to effect suchtreatment or prevention of the disease.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

“Prodrug” means a compound that is convertible in vivo metabolicallyinto an inhibitor according to the present invention. The prodrug itselfmay or may not also have activity with respect to a given targetprotein. For example, a compound comprising a hydroxy group may beadministered as an ester that is converted by hydrolysis in vivo to thehydroxy compound. Suitable esters that may be converted in vivo intohydroxy compounds include acetates, citrates, lactates, phosphates,tartrates, malonates, oxalates, salicylates, propionates, succinates,fumarates, maleates, methylene-bis-β-hydroxynaphthoate, gentisates,isethionates, di-p-toluoyltartrates, methanesulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexylsulfamates, quinates, esters of amino acids, and the like.Similarly, a compound comprising an amine group may be administered asan amide that is converted by hydrolysis in vivo to the amine compound.

An “excipient” is a pharmaceutically acceptable substance formulatedalong with the active ingredient(s) of a medication, pharmaceuticalcomposition, formulation, or drug delivery system. Excipients may beused, for example, to stabilize the composition, to bulk up thecomposition (thus often referred to as “bulking agents,” “fillers,” or“diluents” when used for this purpose), or to confer a therapeuticenhancement on the active ingredient in the final dosage form, such asfacilitating drug absorption, reducing viscosity, or enhancingsolubility. Excipients include pharmaceutically acceptable versions ofantiadherents, binders, coatings, colors, disintegrants, flavors,glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles.The main excipient that serves as a medium for conveying the activeingredient is usually called the vehicle. Excipients may also be used inthe manufacturing process, for example, to aid in the handling of theactive substance, such as by facilitating powder flowability ornon-stick properties, in addition to aiding in vitro stability such asprevention of denaturation or aggregation over the expected shelf life.The suitability of an excipient will typically vary depending on theroute of administration, the dosage form, the active ingredient, as wellas other factors.

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

The term “eflornithine” when used by itself refers to2,5-diamino-2-(difluoromethyl)pentanoic acid is any of its forms,including non-salt and salt forms (e.g., eflornithine HCl), anhydrousand hydrate forms of non-salt and salt forms (e.g., eflornithinehydrochloride monohydrate), solvates of non-salt and salts forms, itsenantiomers (R and S forms, which may also by identified as d and 1forms), and mixtures of these enantiomers (e.g., racemic mixture, ormixtures enriched in one of the enantiomers relative to the other).Specific forms of eflornithine include eflornithine hydrochloridemonohydrate (i.e., CAS ID: 96020-91-6; MW: 236.65), eflornithinehydrochloride (i.e., CAS ID: 68278-23-9; MW: 218.63), and freeeflornithine (i.e., CAS ID: 70052-12-9; MW: 182.17). Where necessary,the form of eflornithine has been further specified. In someembodiments, the eflornithine of the present disclosure is eflornithinehydrochloride monohydrate (i.e., CAS ID: 96020-91-6). The terms“eflornithine” and “DFMO” are used interchangeably herein. Othersynonyms of eflornithine and DFMO include: α-difluoromethylornithine,2-(Difluoromethyl)-DL-ornithine, 2-(Difluoromethyl)ornithine,DL-α-difluoromethylornithine, N-Difluoromethylornithine, ornidyl,αδ-Diamino-α-(difluoromethyl)valeric acid, and2,5-diamino-2(difluro)pentanoic acid.

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis therefore well below 0.05%, preferably below 0.01%. Most preferred isa composition in which no amount of the specified component can bedetected with standard analytical methods.

The term “fixed dose combination” or “FDC” refers to a combination ofdefined doses of two drugs or active ingredients presented in a singledosage unit (e.g., a tablet or a capsule) and administered as such;further as used herein, “free dose combination” refers to a combinationof two drugs or active ingredients administered simultaneously but astwo distinct dosage units.

“Granulation” refers to the process of agglomerating powder particlesinto larger granules that contain the active pharmaceutical ingredient.“Dry granulation” refers to any process comprising the steps where thereis no addition of a liquid to powdered starting materials, agitation,and drying to yield a solid dosage form. The resulting granulated drugproduct may be further processed into various final dosage forms, e.g.,capsules, tablets, wafers, gels, lozenges, etc.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human patients are adults, juveniles, infants and fetuses.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

A “pharmaceutically acceptable carrier,” “drug carrier,” or simply“carrier” is a pharmaceutically acceptable substance formulated alongwith the active ingredient medication that is involved in carrying,delivering and/or transporting a chemical agent. Drug carriers may beused to improve the delivery and the effectiveness of drugs, includingfor example, controlled-release technology to modulate drugbioavailability, decrease drug metabolism, and/or reduce drug toxicity.Some drug carriers may increase the effectiveness of drug delivery tothe specific target sites. Examples of carriers include: liposomes,microspheres (e.g., made of poly(lactic-co-glycolic) acid), albuminmicrospheres, synthetic polymers, nanofibers, nanotubes, protein-DNAcomplexes, protein conjugates, erythrocytes, virosomes, and dendrimers.

The term “physically separated” as defined herein refers to apharmaceutical oral fixed dose combination containing both components a)and b) formulated such that they are not mixed with each other in thesame carrier but are separated. This separation helps to minimize theinteractions between the two components especially upon release of same.Typically the physical separation means that the two components a) andb) are present in different compartments, such as layers, or are presentas different entities, such as particulates or granulates, of theformulation. It is not necessary that the two components a) and b) arefurther separated by additional layers or coating although this may beappropriate from case to case. This physical separation of the twocomponents a) and b) in one dosage form can be achieved by various meansknown in the art. In one embodiment, this is achieved by formulating therespective components a) and b) into separate layers, e.g., a multi- orbilayer formulation. Specific examples of such formulation techniquesare described herein.

The term “sticking” refers to the attachment of granules to the faces oftablet press punches including within the letter, logo or design on thepunch faces.

The term “capping” refers to the split or fracture of the cap or top ofa tablet from the body of the tablet. Capping can be caused bynon-compressible fine particles that migrate when the air is pushed outduring compression.

The term “friability” refers herein to the tendency of a tablet to chip,crumble or break following compression. It can be caused by a number offactors including poor tablet design (too sharp edges), low moisturecontent, insufficient binder, etc. In some aspects, the friability of atablet sample is given in terms of % weight loss (i.e., loss in weightexpressed as a percentage of the original sample weight). Generally, amaximum weight loss of not more than 1% is considered acceptable formost tablets.

The term “release” as used herein refers to a process by which thepharmaceutical oral fixed dose combination is brought into contact witha fluid and the fluid transports the drug(s) outside the dosage forminto the fluid that surrounds the dosage form. The combination ofdelivery rate and delivery duration exhibited by a given dosage form ina patient can be described as its in vivo release profile. The releaseprofiles of dosage forms may exhibit different rates and durations ofrelease and may be continuous. Continuous release profiles includerelease profiles in which one or more active ingredients are releasedcontinuously, either at a constant or variable rate. When two or morecomponents that have different release profiles are combined in onedosage form, the resulting individual release profiles of the twocomponents may be the same or different compared to a dosage form havingonly one of the components. Thus, the two components can affect eachother's release profile leading to a different release profile for eachindividual component.

A two-component dosage form can exhibit release profiles of the twocomponents that are identical or different to each other. The releaseprofile of a two-component dosage form where each component has adifferent release profile may be described as “asynchronous”. Such arelease profile encompasses both (1) different continuous releases wherepreferably component b) is released at a slower rate than component a),and (2) a profile where one of components a) and b), preferablycomponent b), is released continuous and the other of components a) andb), preferably component a), is modified to be released continuous witha time delay. Also a combination of two release profiles for one drug ispossible (e.g. 50% of the drug in continuous and 50% of the same drugcontinuous with a time delay).

Immediate release: For the purposes of the present application, animmediate release formulation is a formulation showing a release of theactive substance(s), which is not deliberately modified by a specialformulation design or manufacturing method.

Modified release: For the purposes of the present application, amodified release formulation is a formulation showing a release of theactive substance(s), which is deliberately modified by a specialformulation design or manufacturing method. This modified release can betypically obtained by delaying the time of release of one or both of thecomponents, preferably component a). Typically for the purposes of thepresent invention, a modified release refers to a release over 5 h, suchas a release over 3 h or even shorter. Modified release as used hereinis meant to encompass both a different continuous release over time ofthe two components or a delayed release where one of the components,preferably component a), is released only after a lag time. Such amodified release form may be produced by applying release-modifyingcoatings, e.g. a diffusion coating, to the drug substance(s) or to acore containing the drug substance(s), or by creating arelease-modifying matrix embedding the drug substance(s).

The term “tablet” refers to a pharmacological composition in the form ofa small, essentially solid pellet of any shape. Tablet shapes maybecylindrical, spherical, rectangular, capsular or irregular. The term“tablet composition” refers to the substances included in a tablet. A“tablet composition constituent” or “tablet constituent” refers to acompound or substance which is included in a tablet composition. Thesecan include, but are not limited to, the active and any excipients inaddition to the low melting compound and the water soluble excipient.

The above definitions supersede any conflicting definition in any of thereference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the invention in terms such that oneof ordinary skill can appreciate the scope and practice the presentinvention.

Unit abbreviations used herein include average result (ar), kilopond(kp), kilonewton (kN), percent weight per weight (% w/w), pounds persquare inch (psi), RH (relative humidity), color difference delta E(dE), and revolutions per minute (rpm).

VIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Development of Eflornithine HCl and Sulindac CombinationTables

In the development process of a fixed dose combination (FDC) tabletcomprising eflornithine HCl and sulindac, several formulations weretested (Table 1). The parameters that were tested included tabletdisintegration time, tablet hardness, and percentage of tabletfriability.

Formulation I was manufactured into a 900 mg tablet by first mixing ⅓ ofthe silicified MCC (PROSOLV®) with the eflornithine HCl in a 1 quartv-blender. Next, the sulindac and ⅓ of the silicified MCC (PROSOLV®) waspre-mixed in a polyethylene (PE) bag and added to the blender along withthe colloidal silicon dioxide (CARBOSIL®) and the pregelatinized cornstarch (STARCH 1500®). The PE bag was rinsed with the remaining ⅓ ofsilicified MCC (PROSOLV®) and added to the blender. The mix was blendedfor 10 minutes at about 25 rpm before the addition of hand screenedmagnesium stearate and then blended for an additional 3 minutes. Thisformulation was found to have some sticking on the punch surface andresulted in a rough tablet surface. Thus, for Formulation II themagnesium stearate was increased from 0.5% to 1% and silicified MCC wasdecreased from 38.57% to 38.07%.

Formulation II was manufactured into a 900 mg tablet by pre-mixingCARBOSIL®, STARCH 1500®, and sulindac in a PE bag. Next, ½ of thePROSOLV® and the eflornithine HCl was added to the 8-quart v-blenderalong with the pre-mix. The remaining ½ of the PROSOLV® was used torinse the PE bag and added to the blender. The mix was blended for 10minutes at about 25 rpm. The mix was then removed from the blender anddelumped through a Comill 039R screen before returning to the v-blenderfor an additional 10 minutes of blending. Next, magnesium stearatehand-screened through a 30 mesh (i.e., 590 μm) screen was added to thev-blender by manual mixing and the mix was blended for 3 minutes atabout 25 rpm. The mix was compressed into a tablet on the Key Model BBTS10 station. The resulting tablet was determined to have a disintegrationtime of about 29-32 seconds, a friability of 0.077% at 4 minutes and0.17392% at 8 minutes, and a hardness of about 28 kp (Table 1). Thetablet was then film coated with OPADRY® Yellow (Colorcon) at a percentweight of 2.913 to produce a tablet of 927 mg using an O'Hara Labcoat,12″ pan. The film coated tablets had a hardness of about 36.0-42.1 kpand disintegration time of 1 minute 27 seconds to 1 minute 53 seconds.

Formulation III was manufactured into a 650 mg tablet by pre-mixingCARBOSIL®, part 2 of the PROSOLV® and sulindac in a PE bag. Next, ½ ofpart 1 of the PROSOLV® and eflornithine were added to the 8-quartv-blender with the pre-mix. The remaining ½ of part 1 of the PROS OLV®was used to rinse the PE bag and added to the v-blender. The mix wasblended for 10 minutes at about 25 rpm. The mix was then removed fromthe blender and delumped through a Comill 039R screen before returningto the v-blender for an additional 10 minutes of blending. Next,magnesium stearate hand-screened through a 30 mesh (i.e., 590 μm) screenwas added to the v-blender by manual mixing and the mix was blended for3 minutes at about 25 rpm. The mix was compressed into a tablet on theKey Model BBTS 10 station. The resulting tablet was determined to have adisintegration time of about 51-57 seconds, a friability of0.2607%-0.3373% at 4 minutes and 0.8988%-1.008% at 8 minutes, and ahardness of about 13 kp. The tablet was then film coated with OPADRY®Yellow (Colorcon) at a percent weight of 2.913 to produce a tablet of669.5 mg using an O'Hara Labcoat, 12″ pan. The film coated tablets had ahardness of about 36.0-42.1 kp and disintegration time of 1 minute 27seconds to 1 minute 53 seconds. This formulation had a reduced weightfrom 900 mg to 650 mg and STARCH 1500® was replaced with PROS OLV® toincrease the tablet strength. However, capping was observed during thefriability testing as well as during the film coating process.

Formulation IV was manufactured into a 700 mg tablet using the sameprocess as Formulation III. The resulting tablet was determined to havea disintegration time of 1 minutes 10 seconds to about 1 minutes 34seconds, a friability of 0.1424%-0.1567% at 4 minutes and0.3186%-0.5166% at 8 minutes, and a hardness of about 20 kp. The tabletwas then film coated with OPADRY® Yellow (Colorcon) at a percent weightof 2.913 to produce a tablet of 721 mg using an O'Hara Labcoat, 12″ pan.The film coated tablets had a disintegration time of 1 minute 43 secondsto 2 minutes 7 seconds. In this formulation, the amount of PROSOLV® wasincreased and the table weight increased from 650 mg to 700 mg. Althoughno capping was observed during friability testing, three tablets didhave capping during film coating.

TABLE 1 Formulations I-IV of Eflornithine HCL and Sulindac Fixed DoseCombination Tablets. Formulation I Formulation II Formulation IIIFormulation IV Unit wt Unit wt Unit wt Unit wt Components (mg) % W/W(mg) % W/W (mg) % W/W (mg) % W/W Eflornithine HCl 375 41.67 375 41.67375 57.69 375 53.571 monohydrate racemate Sulindac 75 8.33 75 8.33 7511.54 75 10.714 Silicified MCC (part 1) 347.13 38.57 342.63 38.07 149.523.0 199.6 28.514 Silicified MCC (part 2) 0 0 0 0 41.075 6.32 41.0755.868 Pre Gel Corn Starch 96.12 10.68 96.12 10.68 0 0 0 0 Colloidalsilicon dioxide 2.25 0.25 2.25 0.25 1.625 1.625 1.625 0.232 Magnesiumstearate 4.5 0.5 9 1 7.8 7.8 7.7 1.1 Uncoated Tablet weight 900 100 900100 650 100 700 100 OPADRY ® Yellow 27.0 19.5 21.0 03B92557 CoatedTablet Weight 927.0 669.5 721.0 Tablet Characteristics Formulation IIFormulation IV Compression force NR 85 psi Hardness (kp) ar 28 ar 20Disintegration time ar30 s ar 1 min 30 s Friability (4 min) (%) 0.080.16 Friability (8 min) (%) 0.17 0.52 (one capped tablet)

TABLE 2 Exemplary Formulation of Eflornithine HCL and Sulindac FixedDose Combination Tablet. Components Unit weight (mg) % (w/w)Eflornithine HCl monohydrate racemate 375.00 52.011 Sulindac 75.0010.402 Silicified microcrystalline cellulose 237.87 32.992 Colloidalsilicon dioxide 1.63 0.226 Magnesium stearate 10.50 1.456 Core tabletweight 700.00 OPADRY ® Yellow 21.00 2.913 Film coated tablet weight721.00 100

TABLE 3 Exemplary Tablet Manufacture Parameters. Variable 7107/2 R3bis7107/2 R4 7107/3 7107/5 R2 7107/5 R3 Mixer Turbula Turbula TurbulaTurbula Turbula Mixing Time 70 cycles 70 cycles 70 cycles 70 cycles 70cycles Mg Stearate 1.50% 1.50% 1.50% 1.50% 1.50% Press Korsch KorschKorsch Ronchi Ronchi Tool dimensions 17.5 × 8 17.5 × 8 17 × 9 R6 16.5 ×7 16.5 × 7 Tool Coating chrome/RC02 chrome/RC02 chrome chrome chromeEngraving Top 414C 414C neutral 4141 4141 Engraving Bottom wave logowave logo neutral logo logo Scored no no cleavable cleavable cleavableCompression Force 37 or 30 kN  37 kN  30 kN  37 kN  25 kNPre-Compression Force 2.1 kN 2.5 kN 2.0 kN 3.7 kN 2.5 kN Test ResultsCleavage no no no no no Sticking no no no no no Top Engraving IntensityPass Pass Pass Pass Pass Bottom Engraving Pass Pass Pass Pass PassIntensity Hardness NA 12.80 kp 8.14 kp 18.46 kp 16.62 kp CleavagingAbility NA yes yes no no Disintegration Time NA l min 15″ to 40 sec to 2min 15″ to 1 min 39″ to 1 min 25″ 45 sec 2 min 32″ 1 min 53″ FriabilityAt 4 Minutes NA 0.08% 0.17% 0.21% 0.37% Friability At 30 Minutes NA1.15% 1.19% 1.80% 2.85% Tablets Broken/Cleaved NA no no no no

TABLE 4 Materials used for the formulations described in Example 1.Material Supplier Efllornithine HCl monohydrate Scino Pharm SulindacZACH Silicified microcrystalline cellulose NF EP (MCC) (PROSOLV ®)Starch 1500 (Partially pregelatinized Maize Starch) Colorcon LimitedColloidal silicon dioxide (CARBOSIL ®) IMCD France SAS MagnesiumStearate Mallinkroot-Tyco OPADRY ® Yellow Colorcon Limited Equipment PKblend master V-blender (1 quart and 8 quart) Quadro Comill model 197Swith 0.039″ screen Key Model BBTS 10 station tableting press O'HaraLabcoat 12″ pan, 0.8 mm nozzle

Example 2—Development of Formulation IV

From Example 1, Formulation IV was further tested to determine whichparameters can be altered to prevent capping and sticking. The firstparameter tested was the compression force and the addition of apre-compression force at about 5-15% of the compression force (Table 5).To evaluate the compression and pre-pressure forces for the FormulationIV 700 mg tablet to reach a hardness of about 20 kp, several trials wereperformed. In a first trial, a final blend of the Formulation IV 700 mgtablet was manufactured using Equipment C (Table 9). The manufacturingprocess involved pre-mixing CARBOSIL®, part 2 of the PROSOLV® andsulindac in a PE bag. Next, ½ of part 1 of the PROSOLV® and eflornithinewere added to a 10-quart v-blender with the pre-mix. The remaining ½ ofpart 1 of the PROS OLV® was used to rinse the PE bag and added to thev-blender. The mix was blended for 35 minutes at about 7 rpm. The mixwas then removed from the blender and delumped through a Frewitt TC1501.0 mm screen before returning to the v-blender for an additional 35minutes of blending. Next, magnesium stearate was hand-screened througha 500 μm screen and added to the v-blender by manual mixing for a finalblend of 10 minutes at 7 rpm. The compression step was performed on aCourtoy Modul P tableting press equipped with five 17.5×8 mm engravedand chromium plated punches. The parameters were set in order to obtaina hardness of between 17.0 and 22.5 kp. It was found that withoutpre-pressure, capping was observed. However, the use of a pre-pressureforce increased the hardness and avoided capping (Table 10). Inaddition, the tablets formed with a pre-pressure force were moreresistant against attrition (i.e., lower friability). In addition, the16.5×8 mm punch of the Key BBTS 10 station tableting press used inExample 1 appeared to be more prone to attrition.

TABLE 5 Compression parameters tested for Formulation IV. Initialsetting 7107/01 setting#3 7107/01 setting#2 Punch shape 16.5 × 8 mmsmooth 17.5 × 8 mm engraved 17.5 × 8 mm engraved Compression force 85psi 34 kN 35 kN Pre-pressure force No No Yes (3 kN) Hardness (kp) ar 20ar 13 (*) ar 17 Disintegration time ar 1 min 30 s ar 1 min 20 sec ar 2min Friability (4 min) (%) 0.16 0.07 0.03 Friability (8 mm) (%) 0.52 (1capped tablet) NA NA Friability (10 min) (%) NA 0.27 0.13 Friability (30min) (%) NA 1.79 (1 capped tablet) 0.54 (no capped tablet) Thickness(mm) ar 6.1 ar 5.5 ar 5.4 NA: not applied (*) maximum hardness that canbe reached without precompression.

In a second trial, the punch surface was varied to determine its effecton the Formulation IV tablet (Table 11). The final blend of theFormulation IV 700 mg tablet was manufactured using Equipment B in thistrial. The manufacturing process involved pre-mixing CARBOSIL®, part 2of the PROS OLV® and sulindac in a PE bag. Next, ½ of part 1 of thePROSOLV® and eflornithine were added to a 10-quart v-blender with thepre-mix. The remaining ½ of part 1 of the PROS OLV® was used to rinsethe PE bag and added to the v-blender. The mix was blended for 8 minutes30 seconds at about 30 cycles per minute. The mix was then removed fromthe blender and delumped through a CMA 1.0 mm screen before returning tothe v-blender for an additional 8.5 minutes of blending. Next, magnesiumstearate hand-screened through a 500 μm screen and added to thev-blender by manual mixing for a final blend of 2 minutes 20 seconds at30 cycles per minute. The compression step was performed on a KorschXL100 tableting press equipped with two 17.5×8 mm engraved andanti-sticking chromium plated punches. The pre-pressure was set at 5-10%of the main compression force which was around 30 kN. Several differentpunch surfaces were also tested including chromium, carbon, tungsten,and Teflon VS stainless steel. In some embodiments, Teflon may be usedto reduce the sticking.

To avoid sticking, several additional variables were tested and a highconstraint was applied at the very beginning of the compression. Neitherlubrication with 1.1% magnesium stearate nor increasing the lubricationtime from 70 rotations to 140 rotations prevented sticking (Tables 11and 12). However, increasing the ratio of magnesium stearate to 1.5% didprevent sticking (Table 12) along with a slight decrease in tablethardness at about 20%, but the friability was still very low at lessthan 0.1% after 4 minutes. With two types of punches, equipped withdifferent kinds of break line (17×9 mm and 16.5×7 mm), breakabilityresults were compliant for both punches tested. Thus, increasing themagnesium stearate to 1.5% prevents sticking and pre-compressionprevents capping of Formulation IV.

TABLE 6 Batch weights of Formulation IV in Trial 1 and Trial 2.Components Unit wt (mg) Trial 1 wt (g) Trial 2 wt (g) Eflornithine HCl375 1339.500 1340.000 Sulindac 75 268.100 268.027 Silicified MCC(part 1) 199.6 712.800 712.000 Silicified MCC (part 2) 41.075 146.700146.648 Colloidal silicon dioxide 1.625 5.796 5.8043 Magnesium stearate7.7 27.515 27.504 Tablet weight 700.0 2500.411 2499.983

TABLE 7 Varying Amounts of Magnesium Stearate for Formulation IV. 1.1%of Magnesium 1.3% of Magnesium 1.5% of Magnesium stearate stearateformula stearate formula (*) formula (*) Unit wt Unit wt Unit wtComponents (mg) w/w (%) (mg) w/w (%) (mg) w/w (%) Eflornithine HCl375.000 53.571 374.227 53.461 373.457 53.351 Sulindac 75.000 10.71474.851 10.693 74.704 10.672 Silicified MCC (part 1) 199.598 28.514199.192 28.456 198.793 28.399 Silicified MCC (part 2) 41.075 5.86840.992 5.856 40.908 5.844 Colloidal silicon dioxide 1.625 0.232 1.6240.232 1.617 0.231 Magnesium stearate 7.700 1.100 9.100 1.300 10.5001.500 Tablet weight 700.0 100.00 700.0 100.00 700.0 100.00 (*) Formulaeobtained after dilution to increase the percentage of magnesiumstearate. APIs concentration consequently slightly below the target.

TABLE 8 Coating of Formulation IV. Components Unit wt (mg) Batch wt (g)Uncoated tablets 700.00 600.00 OPADRY ® Yellow 03B92557 21.00 53.995Purified water 154.00 395.99 Coated Tablet weight 721.00 653.995

TABLE 9 Equipment used for development of Formulation IV. Equipment AEquipment B Equipment C PK blend master Turbula T10A Servolift blenderV-blender blender 1 quart and 8 quart 10 L container 10 L containerQuadro Comill 197 S CMA T1 conical Frewitt TC150 0.039″ screen millconical mill 1.00 mm screen 1.00 mm screen 0.500 mm sieving 0.500 mmsieving screen screen Key BBTS 10 station Korsch XL100 Courtoy Modul Ptableting press tableting press tableting press O'Hara Labcoat 12″ panMini Glatt coater

TABLE 10 First trial parameters and results for testing effect ofpre-compression force on Formulation IV. Compression Parameters 7107/01setting#3 7107/01 setting#5 7107/01 setting#2 7107/01 setting#4 Speed(tpm) 50 50 50 50 Pre-pressure force (kN)/ 0.11/0% 1.43/5% 3.25/10%4.68/15% % of the main pressure Compression force (kN) 33.58 32.23 34.0733.03 Punches (quantity) 5 5 5 5 Punch shape 17.5 × 8 mm 17.5 × 8 mm17.5 × 8 mm 17.5 × 8 mm engraved engraved engraved engraved Punchsurface treatment Anti-sticking Anti-sticking Anti-stickingAnti-sticking chromium plating chromium plating chromium platingchromium plating Results Test Sampling 7107/01 setting#3 7107/01setting#5 7107/01 setting#2 7107/01 setting#4 Weight (mg) 20 tablets702.28 699.14 703.5 701.17 RSD (%) 1.18 1.00 1.06 0.73 Hardness (kp) 10tablets 11.5 to 14.4 15.2 to 17.9 15.8 to 18.4 17.0 to 18.7 (Mean value:13.2) (Mean value: 16.6) (Mean value: 17.3) (Mean value: 17.9)Friability (%) According to  4 min Pharmacopeia 0.07/No capping 0.07/Nocapping 0.03/No capping 0.08/No capping 10 min 0.27/No capping 0.20/Nocapping 0.13/No capping 0.19/No capping 30 min 1.79/1 capping 0.67/Nocapping 0.54/No capping 0.59/No capping Disintegration  3 tablets 1 min08 sec 1 min 39 sec 1 min 51 sec 1 min 41 sec time (min) to to to to 1min 40 sec 2 min 17 sec 2 min 11 sec 1 min 57 sec Thickness 10 tablets5.4 to 5.6 5.4 to 5.5 5.4 to 5.5 5.4 to 5.5 (mm) Sticking Some stickingSome sticking Some sticking Some sticking

TABLE 11 Second trial parameters and results for testing effect of punchsurface on Formulation IV. Final blend 7107/02 setting#2 7107/02setting#5 7107/02 setting#6 7107/02 setting#7 7107/02 setting#8 Ratio ofMg stearate 1.1 1.1 1.1 1.1 1.1 (%) Final blend 140 140 140 140 140(rotations) Compression parameters Speed (tpm) 40 40 40 40 40Pre-pressure force 2.5 2.2 2.2 2.1 2.1 (kN) Compression force 30 30 3030 30 (kN) Punches (quantity) 2 2 2 2 2 Punch shape 17.5 × 8 mm 17.5 × 8mm 17.5 × 8 mm 17.5 × 8 mm 17.5 × 8 mm engraved engraved engravedengraved engraved Punch surface Anti-sticking Anti-sticking withAnti-sticking with Anti-sticking with Steel (no anti- treatment chromiumRC-02 carbon RB-01 tungsten RD-03 teflon RF-03 sticking plating) Resultstest sampling 7107/02 setting#2 7107/02 setting#5 7107/02 setting#67107/02 setting#7 7107/02 setting#8 Weight (mg)/RSD 20 tablets697.12/0.38 NA NA NA NA (%) Hardness (kp)  5 tablets 15.4 to 16.3 NA NANA NA Friability (%)  4 min According to 0.02/No capping NA NA NA NA 10min Pharmacopeia 0.04/No capping 30 min 0.69/No capping Disintegrationtime  3 tablets 0 min 58 sec (min) to NA NA NA NA 1 min 00 sec Thickness(mm) 10 tablets 5.5 to 5.5 NA NA NA NA Sticking 10 tablets Some stickingSome sticking Some sticking Very slightly Some sticking sticking

TABLE 12 Second trial parameters and results for testing effect of finalmixing duration and magnesium stearate on Formulation IV. 7107/027107/02 7107/02 7107/02 7107/02 setting#1 setting#2 setting#3 setting#4setting#10 Final blend Ratio of Magnesium 1.1 1.1 1.5 1.5 1.3 stearate(%) Final mixing duration 70 140 70 70 140 (rotations) Compressionparameters Speed (tpm) 40 40 40 40 40 Pre-pressure force 3.5 2.5 2.1 2.52.2 (kN) Compression force >>30 30 30 37 37 (kN) Punches (quantity) 2 22 2 2 Punch shape 17.5 × 8 mm 17.5 × 8 mm 17.5 × 8 mm 17.5 × 8 mm 17.5 ×8 mm engraved engraved engraved engraved engraved Punch surfaceAnti-sticking Anti-sticking Anti-sticking Anti-sticking Anti-stickingtreatment chromium plating chromium plating chromium plating chromiumplating chromium plating Results test sampling Weight (mg)/RSD 20tablets 704.01 (*)/0.23 697.12/0.38 695.19/0.38 702.61/0.39 703.24/0.29(%) Hardness (kp)  5 tablets 17.3 to 17.9 15.4 to 16.3 12.4 to 13.4 11.9to 14.1 13.4 to 14.7 Friability (%)  4 min According to NA 0.02/Nocapping 0.03/No capping 0.08/No capping 0.05/No capping 10 minPharmacopeia NA 0.04/No capping 0.15 No capping 0.11/No capping 0.19/Nocapping 30 min NA 0.69/No capping 1.01/No capping 1.15/No capping1.02/No capping Disintegration time  3 tablets 1 min 15 sec 0 min 58 sec1 min 00 sec 1 min 15 sec 1min30sec (min) to to to to to 1 min 20 sec 1min 00 sec 1 min 10 sec 1 min 25 sec 1 min 45 sec Thickness (mm) 10tablets 5.3 to 5.4 5.5 to 5.5 5.5 to 5.5 5.5 to 5.5 5.5 to 5.6 Sticking10 tablets Decreasing of the Decreasing of the Very slightly No stickingbut Slightly sticking sticking, some sticking sticking on the tendencyto split on the upper lower punches are upper punches. during the punchclean No sticking on the hardness test lower one (*) on 10 tablets

TABLE 13 Trial parameters and results for testing effect of compressionparameters on Formulation IV. 7107/03 setting#1 7107/05 setting#17107/05 setting#2 7107/05 setting#3 Final blend Ratio of Mg stearate (%)1.5 1.5 1.5 1.5 Compression parameters Speed (tpm) 40 40 40 40Pre-pressure force (kN) 2.0 5.0 3.7 2.5 Compression force (kN) 30 24 3725 Punches (quantity) 2 2 2 2 Punch shape 17 × 9R6 mm 16.5 × 7 mm 16.5 ×7 mm 16.5 × 7 mm breakable breakable breakable breakable Punch surfacetreatment Anti-sticking Anti-sticking Anti-sticking Anti-stickingchromium chromium chromium chromium Results test sampling 7107/03setting#1 7107/05 setting#1 7107/05 setting#2 7107/05 setting#3 Weight(mg)/RSD (%) 20 tablets 700.20 (*)/0.49 705.29/0.59 709.62/0.64700.08/0.54 Breaking test RSD on one half 30 tablets 0.97 NA 3.19 2.91(%) Hardness (kp)  5 tablets 7.6 to 8.8 (**) 18.6 to 19.7 17.3 to 19.316.1 to 16.9 Friability (%)  4 min According to 0.17/No capping 0.12/Nocapping 0.21/No capping 0.37/No capping 10 min Pharmacopeia 0.32/Nocapping 0.47/No capping 0.51/No capping 1.04/No capping 30 min 1.19/Nocapping 1.52/No capping 1.80/No capping 2.85/No capping Disintegrationtime (min)  3 tablets 0 min 40 sec 1 min 38 sec 2 min 15 sec 1 min 39sec to to to to 0 min 45 sec 1 min 43 sec 2 min 32 sec 1 min 53 secThickness (mm) 10 tablets 5.3 to 5.3 6.6 to 6.7 6.5 to 6.7 6.6 to 6.7Sticking 10 tablets No sticking No sticking No sticking No sticking (*)on 30 tablets (**) on 10 tablets NA: not applied

The stability of the Formulation IV combination tablet, eflornithinesingle tablet and sulindac single tablet was tested. Stability analysisof the Formulation IV tablets was performed at 6 months using the KarlFischer titration method for determination of water content (FIG. 1). InFIG. 1, it is shown that the combination tablet of Formulation IV had alower uptake of water over six months as compared to the eflornithinesingle tablet. Water can affect drug potency and drug dissolution; forexample, water can increase the rate of drug degradation by hydrolysis(Gerhardt, 2009). Thus, in some embodiments, the combination tabletsprovided herein are more stable than one or both of the single activeagent tablets.

Finally, the dissolution profile of Formulation IV was also tested. Thedissolution study was carried out in 50 mM sodium phosphate buffermedium at a pH of 7.2 using a paddle stirring element at 75 rpm (USP<711> Dissolution Apparatus II (Paddle)) (FIGS. 2A-2B). The method wasvalidated level II for the dissolution of elfonithine and sulindac. Nointerference of active pharmaceutical ingredients eflornithine andsulindac were observed between themselves, with the dissolution medium,with the phosphate buffer solution, or with the excipients.Surprisingly, the fixed dose combination of Formulation IV was observedto have an overlapping in vitro dissolution profile as compared to thesingle agent tablets.

Example 3—Drug Excipient and Coating Compatibility

A non-cGMP drug excipient compatibility study for eflornithineHCl/sulindac combination tablet was conducted. Appearance, HPLC Assayand XRPD properties were evaluated using a series of samples. Theexcipients that were tested included PVP, HPMC, lactose, EXPLOTAB™,Ac-Di-Sol®, PROSOLV®, STARCH 1500®, and OPADRY® Yellow. Samples preparedfor the excipient compatibility were all 1:1 physical mixtures of API(s)with excipient(s), except the eflornithine HCl:sulindac preparation thatwas 5:1, and the eflornithine HCl:sulindac:H₂O preparation that wasabout 6:1:0.3. Total mass of most samples was approximately 750 mg.Preparation involved weigh off of components into 20 cc scintillationvials, closed and vortexed for approximately 30 seconds. The sampleswere then stored in a 40° C./75% RH stability chamber for four weeks.Lids on the vials were loosely secured and were protected from lightwhile stored in the chamber.

Appearance observations were conducted by visual examination of thevials prepared for HPLC analysis. Excipient compatibility samples wereextracted with 50% acetonitrile in buffer (50 mM phosphate buffer pH2.55). Samples containing only Sulindac were prepared by weighing outportion (˜150 mg) of the sample and extracted in a pre-determined volumesuch that the final concentration of eflornithine and sulindac is 9.5mg/mL and 0.1 mg/mL, respectively. The rest of the compatibility sampleswere prepared by quantitative transfer using the extraction solvent in apre-determined volume such that the final concentration of eflornithineand sulindac was approximately the same as above. Excipientcompatibility samples were analyzed using a method capable of detectingboth actives, eflornithine and sulindac (FIG. 4A). The method employs agradient reverse phase HPLC with Ultraviolet (UV) detection at 195 nm.

XRPD analysis was conducted on a Bruker AXS D8 Advance system with aBragg-Brentano configuration using the CuKα radiation. Samples wereanalyzed at room temperature using the following parameters: 40 kV, 40mA, 1° divergence and antiscatter slits, a method measuring incontinuous mode from 2−40° 2Θ with a 0.05° step and 1 second/step time.Between 3 and 25 mg of sample was analyzed using a rotating, top-filledsteel sample holder in a nine-position auto-sampler accessory. Thesystem was calibrated using traceable standards. Results are shown inFIGS. 4B-4C.

Eflornithine HCl with PVP K30 showed moisture in the sample starting inthe 2 week sample and becoming a liquid at 4 weeks. Sulindac with PVPK30showed sticking of the sample at 2 weeks and continuing at 4 weeks.PVPK30 excipient only showed moisture starting in the 2 week sample andbecoming a liquid at 4 weeks. The same behavior was observed with theEflornithine HCl samples but not with the Sulindac samples. HPLC Assayresults for the majority of the samples tested show no distinctive trend(increasing or decreasing) over the different time points. Although anumber of samples had unusually low assay values, the assay levelsshowed more of an increasing trend or remain relatively constant overthe 4 weeks period. The highest variability in assay results wasobserved for the Sulindac/Eflornithine ProSolv SMCC90 sample. The assayvalue at the 4-week time point was 10.0% higher than the assay resultsat initial. This variability may be contributed to the method(nonvalidated) and sample consistency at the different time points.While the acceptable random analytical error of a validated method is2%, the variability of this method is unknown. Except for some of thesamples, the assay values in each of the samples tested over thedifferent time points are within the normally acceptable 2% random errorof an analytical method. There is no distinctive trend for the API,eflornithine and sulindac under the stressed conditions tested. Theresults of this study suggest that both APIs (eflornithine HCl/sulindac)were compatible with the potential excipients.

The drug excipient compatibility study was conducted by XRPD analysis todetermine the crystallinity of the API(s) with potential formulationexcipients for eflornithine HCl/sulindac combination product. The XRPDresults showed no interaction between the API(s) and excipient at 40°C./75% RH after four weeks. This indicated that both APIs (eflornithineHCl/sulindac) were compatible with the potential excipients.

Coating trials were carried out on tablets to determine effect onstability at 1 month and 3 months at a moisture content of 25° C./60% RHor 40° C./75% RH. The coatings included OPADRY® Yellow (Colorcon,03B92557), OPADRY® White (Colorcon Y-1-7000), OPADRY® II White (Colorcon85F18422), and OPADRY® Clear (Colorcon YS-3-7413) at a 3 percent or 4percent weight gain. The color eye measurement was taken to evaluate thetotal color difference, or DE, between the tablets that were onstability and the initial coated tablets.

The tablet color was tested using a Datacolor Spectraflash 600 SeriesSpectrophotometer. The data was analyzed using the CommissionInternationale de l'Eclairage (CIE) L*a*b* system. In the L*a*b* systemcolor is represented as a coordinate in a three dimensional space.Lightness and darkness are plotted on the L* axis with L=100representing pure white and L=0 representing pure black. The a* and b*axes represent the two complementary color pairs of red/green andblue/yellow respectively. By plotting colors geometrically thedifference between two colors (total color difference=E*) can bedetermined by calculating the distance between two points using thefollowing equation.

DE*=[(L*1−L*2)2+(a*1−a*2)2+(b*1−b*2)2]½

Using the Datacolor, each tablet was analyzed at each weight gain of thevarious coating formulations. The closer the DE value is to zero, thecloser the tested tablet color is to the color standard (the initialsamples). Colorcon's standard spec for white coatings (to pass QCtesting) would be a DE value of less than 1.5. All stability sampleswith white film coating exceed that 1.5 DE and therefore would not passColorcon's standard QC testing (Table 14). The clear coated tablets werealso well above the value of 1.5.

TABLE 14 DE values for coated tablets on stability. 3% wg 4% wg 3% wg 4%wg 3% wg 4% wg 3% wg Y-1-7000 Y-1-7000 85F18422 85F18422 03B9255703B92557 YS-3-7413 (white) (white) (white) (white) (yellow) (yellow)(clear) 1 mo 25/60 1.81 1.64 2.56 2.8 0.27 0.32 1.15 3 mo 25/60 1.971.94 2.96 2.31 0.35 0.22 1.1 1 mo 40/75 1.91 2.47 3.58 2.39 0.3 0.294.29 3 mo 40/75 2.71 2.66 2.72 3.31 0.64 0.58 7.6

The best DE results were seen with the tablets coated with the yellowformulation. The DE values were well below 1.5. A DE value (total colordifference) of 1 or below is considered imperceptible to the human eye.Colorcon's typical internal specification for yellow coatings tend to bea DE value of 2.5-3. Thus, OPADRY® Yellow was used to coat thecombination tablets.

Example 4—Bioequivalence Study of Fixed Co-FormulatedEflornithine/Sulindac

A pilot study was performed to compare the pharmacokinetic parameters ofeflornithine, sulindac, sulindac sulfide, and sulindac sulfone in plasmafollowing oral administration of the co-formulated tablet containingeflornithine/sulindac compared to individual tablets containingeflornithine or sulindac taken alone or co-administered in normalhealthy subjects under fasting conditions. The secondary objective ofthis study was to determine the safety and tolerability ofeflornithine/sulindac co-formulated tablets compared to individualformulations taken alone or co-administered in normal healthy subjects.

The study comprised twelve subjects, male or female, at least 18 yearsof age but not older than 60 years. The main inclusion criteria were:light-, non- or ex-smokers; body mass index (BMI)≥18.50 kg/m² and <30.00kg/m²; no clinically significant abnormality found in the 12-lead ECGperformed (subjects had to be in a supine position for 10 minutes priorto ECG, and the ECG was performed prior to all requested blood draws);negative pregnancy test for female subjects; and healthy according tomedical history, complete physical examination (including vital signs)and laboratory tests (general biochemistry, hematology and urinalysis).

The subjects were treated in four treatment groups comprising:

-   -   Treatment 1: a single 750/150 mg dose of co-formulated        Eflornithine 375 mg/Sulindac 75 mg tablets (2×375/75 mg tablets)    -   Treatment 2: a single 750 mg dose of Eflornithine 250 mg tablets        (3×250 mg tablets)    -   Treatment 3: a single 150 mg dose Sulindac 150 mg tablets (1×150        mg tablet)    -   Treatment 4: a single 150 mg dose of Sulindac 150 mg tablets        (1×150 mg tablet) and a single 750 mg dose of Eflornithine 250        mg tablets (3×250 mg tablets) administered concurrently

Each subject was assigned to receive the 4 different treatments over a28-day period. A single oral dose of the assigned treatment wasadministered under fasting conditions in each study period. Thetreatment administrations were separated by a wash-out of 7 calendardays. A total of 120 blood samples were collected in 80 occasions foreach subject. The first blood sample was collected prior to drugadministration while the others were collected 0.25, 0.5, 0.75, 1, 1.5,2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 16, 24, 36 and 48 hours post drugadministration. The analytes were measured by HPLC with MS/MS detection.The assay range was 35.0 ng/mL to 35000.0 ng/mL for eflornithine, 30.0ng/mL to 15000.0 ng/mL for sulindac, and 10.0 ng/mL to 8000.0 ng/mL forsulindac sulfone and sulindac sulfide. Safety was evaluated throughassessment of adverse events (AEs), standard laboratory evaluations,vital signs, and ECGs.

Mathematical Model and Statistical Methods of PharmacokineticParameters: The main absorption and disposition parameters werecalculated using a non-compartmental approach with a log-linear terminalphase assumption. The trapezoidal rule was used to estimate area underthe curve. The terminal phase estimation was based on maximizing thecoefficient of determination. The pharmacokinetic parameters of thistrial were C_(max), T_(max), AUC_(0-T), AUC_(0-∞), AUC_(0-T/∞), λ_(Z)and T_(half). The statistical analysis was based on a parametric ANOVAmodel of the pharmacokinetic parameters; the two-sided 90% confidenceinterval of the ratio of geometric means for the C_(max), AUC_(0-T) andAUC_(0-∞) was based on ln-transformed data; the T_(max) wasrank-transformed. The ANOVA model used fixed factors of sequence,period, and treatment; the random factor was subject nested withinsequence.

The pharmacokinetic parameters included C_(max) (Maximum observed plasmaconcentration), T_(max) (Time of maximum observed plasma concentration;if it occurs at more than one time point, T_(max) is defined as thefirst time point with this value), T_(LQC) (Time of last observedquantifiable plasma concentration), AUC_(0-T) (Cumulative area under theplasma concentration time curve calculated from 0 to T_(LQC) using thelinear trapezoidal method), AUC_(0-∞) (Area under the plasmaconcentration time curve extrapolated to infinity, calculated asAUC_(0-T)+C_(LQC)/λ_(Z), where C_(LQC) is the estimated concentration attime T_(LQC)), AUC_(0-T/∞), (Relative percentage of AUC_(0-T) withrespect to AUC_(0-∞)), T_(LIN) (Time point where log-linear eliminationphase begins), λ_(Z) (Apparent elimination rate constant, estimated bylinear regression of the terminal linear portion of the logconcentration versus time curve), and T_(half) (Terminal eliminationhalf-life, calculated as ln(2)/λ_(Z)).

TABLE 15 Pharmacokinetic Parameters for Eflornithine Treatment-1 (n =12) Treatment-2 (n = 12) Treatment-4 (n = 12) PARAMETER MEAN C.V. (%)MEAN C.V. (%) MEAN C.V. (%) C_(max) (ng/mL) 10643.8 (21.6) 10234.6(19.9) 10012.8 (25.5) In (C_(max)) 9.2525 (2.2) 9.2134 (2.3) 9.1822(2.8) T_(max) (hours)* 3.25 (2.00-6.00) 3.50 (2.00-5.00) 4.50(2.50-5.00) AUC_(0-T) (ng · h/mL) 71459.8 (20.4) 68962.3 (20.2) 69914.9(18.3) In (AUC_(0-T)) 11.1562 (1.9) 11.1229 (1.8) 11.1407 (1.6)AUC_(0-∞) (ng · h/mL) 71839.3 (20.3) 69301.2 (20.0) 70326.0 (18.1) In(AUC_(0-∞)) 11.1619 (1.9) 11.1281 (1.8) 11.1468 (1.6) AUC_(0-T/∞) (%)99.44 (0.3) 99.48 (0.2) 99.39 (0.3) λ_(z) (hours⁻¹) 0.1453 (25.0) 0.1642(21.5) 0.1630 (26.3) T_(half) (hours) 5.07 (27.3) 4.43 (24.9) 4.65(39.0) *Median (range)

TABLE 16 Pharmacokinetic Parameters for Sulindac Treatment-1 Treatment-3Treatment-4 (n = 12)** (n = 12)** (n = 12)*** PARAMETER MEAN C.V. (%)MEAN C.V. (%) MEAN C.V. (%) C_(max) (ng/mL) 4553.4 (31.6) 5236.1 (39.2)5188.5 (42.9) In (C_(max)) 8.3788 (3.7) 8.4946 (4.7) 8.4562 (5.7)T_(max) (hours)* 1.54 (0.75-5.00) 1.50 (1.00-2.50) 1.50 (0.75-5.00)AUC_(0-T) (ng · h/mL) 11268.3 (32.2) 11569.7 (31.4) 11340.8 (43.9) In(AUC_(0-T)) 9.2823 (3.5) 9.3114 (3.4) 9.2621 (4.2) AUC_(0-∞) (ng · h/mL)11579.4 (39.9) 12687.8 (34.9) 12023.7 (49.3) In (AUC_(0-∞)) 9.2896 (4.2)9.3924 (3.9) 9.3019 (4.8) AUC_(0-T/∞) (%) 96.73 (4.9) 98.14 (1.2) 97.58(1.6) λ_(z) (hours⁻¹) 0.2810 (48.0) 0.3408 (45.9) 0.2034 (58.0) T_(half)(hours) 4.97 (142.9) 2.88 (83.5) 4.61 (55.3) *Median (range) **n = 7 forAUC_(0-∞), λ_(z) and T_(half) ***n = 8 for AUC_(0-∞), λ_(z) and T_(half)

Criteria for Bioequivalence: Statistical inference of eflornithine wasto be based on a bioequivalence approach using the ratio of geometricLSmeans with corresponding 90% confidence interval calculated from theexponential of the difference between Treatment 1 vs Treatment 2,Treatment 2 vs Treatment 4 and Treatment 1 vs Treatment 4 for theln-transformed parameters C_(max), AUC_(0-T) and AUC_(0-∞) were all tobe compared to the 80.00 to 125.00% range. Statistical inference ofsulindac was to be based on a bioequivalence approach using the ratio ofgeometric LSmeans with corresponding 90% confidence interval calculatedfrom the exponential of the difference between Treatment 1 vs Treatment3, Treatment 3 vs Treatment 4 and Treatment 1 vs Treatment 4 for theln-transformed parameters C_(max), AUC_(0-T) and AUC_(0-∞) were all tobe compared to the 80.00 to 125.00% range. The same criteria were to beapplied for sulindac sulfide and sulindac sulfone and the results wereto be presented as supportive evidence of comparable therapeuticoutcome.

Safety results: A total of 12 subjects entered the study, and allsubjects received the 4 treatments under study. No serious adverseevents (SAE) and no deaths were reported for any of the subjectsenrolled in this study. No subject was withdrawn by the investigator forsafety reasons. A total of 4 treatment-emergent adverse events (TEAEs)were reported by 4 (33%) of the 12 subjects who participated in thisstudy. Of these events, 2 occurred after administration Treatment 1, 1after administration of Treatment 3, and the remaining one afteradministration of Treatment 4. Subjects dosed with Treatment 2 did notreport any TEAEs. Half of the TEAEs experienced during the study wereconsidered related to drug administration.

The TEAEs in this study were experienced with a low incidence; they wereexperienced by 1 subject (8%) per treatment group. Dry mouth wasreported following administration of Treatment 4, upper respiratorytract infection was reported following administration of Treatment 3,and vessel puncture site bruise and headache were each reportedfollowing administration of Treatment 1.

The incidence of TEAEs was the same for subjects dosed with Treatment 3and Treatment 4 (8%) and slightly lower than the one reported forsubjects dosed with Treatment 1 (17%). Drug-related TEAEs were reportedwith the same incidence for subjects dosed with Treatment 1 andTreatment 4 (8%), whereas subjects dosed with Treatment-3 did notexperience drug-related TEAEs. The TEAEs experienced during the studywere deemed mild (¾, 75%) and moderate (¼, 25%) in intensity. None ofthe subjects experienced a severe TEAE during the study.

All the abnormal clinical laboratory values were marginally higher orlower than their reference ranges and none were considered clinicallysignificant by the investigator. Furthermore, there were no clinicallysignificant abnormalities in the vital signs and ECGs of the subjects inthis study. All physical examinations were judged normal. Overall, thedrugs tested were generally safe and well tolerated by the subjectsincluded in this study.

Eflornithine Comparison between Treatment 1 and Treatment 2: Thepharmacokinetic results demonstrate that the geometric LSmean ratios andthe corresponding 90% confidence intervals of C_(max), AUC_(0-T), andAUC_(0-∞) of eflornithine were all included within the range of 80.00%to 125.00%. The results of this comparison indicate that bioequivalencecriteria were met when Treatment 1 and Treatment 2 were administeredunder fasting conditions and demonstrate that eflornithinebioavailability is comparable between the co-formulated tabletcontaining eflornithine/sulindac and the tablet containing eflornithinealone.

TABLE 17 Summary of Statistical Analysis of Eflornithine in Treatment 1vs. Treatment 2 INTRA- SUBJECT GEOMETRIC LSMEANS * 90% CONFIDENCE C.V.Treatment-1 Treatment-2 RATIO LIMITS (%) PARAMETER (%) (n = 12) (n = 12)(%) LOWER UPPER C_(max) 16.8 10430.9 10030.8 103.99 92.42 117.01AUC_(0-T) 13.5 69998.7 67701.4 103.39 94.03 113.69 AUC_(0-∞) 13.470395.4 68056.2 103.44 94.17 113.61 * units are ng/mL for C_(max) and ng· h/mL for AUC_(0-T) and AUC_(0-∞)

Eflornithine Comparison between Treatment 2 and Treatment 4: Thepharmacokinetic results demonstrate that the geometric LSmean ratios andthe corresponding 90% confidence intervals of C_(max), AUC_(0-T), andAUC_(0-∞) of eflornithine were all included within the range of 80.00%to 125.00%. The results of this comparison indicate that bioequivalencecriteria were met when Treatment 2 and Treatment 4 were administeredunder fasted conditions and demonstrate that co-administration ofsulindac with the individual tablet of eflornithine did not influencethe bioavailability of eflornithine when administered alone.

TABLE 18 Summary of Statistical Analysis of Eflornithine in Treatment 2vs. Treatment 4 GEOMETRIC LSMEANS * INTRA- Treatment- Treatment- 90%CONFIDENCE SUBJECT 2 4 RATIO LIMITS (%) PARAMETER C.V. (%) (n = 12) (n =12) (%) LOWER UPPER C_(max) 16.8 10030.8 9722.7 103.17 91.69 116.09AUC_(0-T) 13.5 67701.4 68916.4 98.24 89.34 108.02 AUC_(0-∞) 13.4 68056.269338.0 98.15 89.36 107.81 * units are ng/mL for C_(max) and ng · h/mLfor AUC_(0-T) and AUC_(0-∞)

Eflornithine Comparison between Treatment 1 and Treatment 4: Thepharmacokinetic results demonstrate that the geometric LSmean ratios andthe corresponding 90% confidence intervals of C_(max), AUC_(0-T), andAUC_(0-∞) of eflornithine were all included within the range of 80.00%to 125.00%. The results of this comparison indicate that bioequivalencecriteria were met when Treatment 1 and Treatment 4 were administeredunder fasted conditions and demonstrate that the bioavailability ofeflornithine for the co-formulated tablet containingeflornithine/sulindac and the co-administration of individual tabletscontaining each eflornithine or sulindac is similar.

TABLE 19 Summary of Statistical Analysis of Eflornithine in Treatment 1vs. Treatment 4 INTRA- GEOMETRIC LSMEANS * 90% CONFIDENCE SUBJECTTreatment-1 Treatment-4 RATIO LIMITS (%) PARAMETER C.V. (%) (n = 12) (n= 12) (%) LOWER UPPER C_(max) 16.8 10030.8 9722.7 107.28 95.35 120.72AUC_(0-T) 13.5 67701.4 68916.4 101.57 92.37 111.68 AUC_(0-∞) 13.468056.2 69338.0 101.53 92.43 111.51 * units are ng/mL for C_(max) and ng· h/mL for AUC_(0-T) and AUC_(0-∞)

Sulindac Comparison between Treatment 1 between Treatment 3: Thepharmacokinetic results demonstrate that the geometric LSmean ratios andthe corresponding 90% confidence intervals (90 CI) of C_(max),AUC_(0-T), and AUC_(0-∞) of sulindac were not all included within therange of 80.00% to 125.00%. The lower bound of the 90 CI of C_(max) wasbelow the 80.00% limit. Since the ratios were within the 80.00% to125.00% range for all PK parameters, the intra-subject variability couldaccount for the lower bound of C_(max) being outside the BE range. Theresults obtained for this comparison demonstrate that the sample sizeused in this pilot study was not sufficient to demonstrate equivalenceof sulindac bioavailability from the co-formulated tablet and sulindacalone.

TABLE 20 Summary of the Statistical Analysis of Sulindac in Treatment 1vs Treatment 3 INTRA- GEOMETRIC LSMEANS * 90% CONFIDENCE SUBJECTTreatment-1 Treatment-3 RATIO LIMITS (%) PARAMETER C.V. (%) (n = 12) (n= 12) (%) LOWER UPPER C_(max) 24.6 4353.6 4888.5 89.06 75.04 105.69AUC_(0-T) 11.9 10746.4 11063.6 97.13 89.34 105.60 AUC_(0-∞) 13.6 12029.412743.6 94.40 82.27 108.30 * units are ng/mL for C_(max) and ng · h/mLfor AUC_(0-T) and AUC_(0-∞) **n = 7 for AUC_(0-∞)

Based on the data, the intra-subject variation, that incorporates thevariability between all comparisons, is about 24.6% for C_(max) andabout 12% for AUC_(0-T). Statistically, given that the expectedTreatment 1 to Treatment 3 ratio of geometric LSmeans felt within 90 and110%, it is estimated that the number of subjects to meet the 80.00 to125.00% bioequivalence range with a statistical a priori power of atleast 80% would be about 54 for a future pivotal study. The inclusion of60 subjects should be sufficient to account for the possibility ofdrop-outs and variations around the estimated intra-subject CV.

Sulindac Comparison between Treatment 3 and Treatment 4: Thepharmacokinetic results demonstrate that the geometric LSmean ratios andthe corresponding 90% confidence intervals of C_(max), AUC_(0-T), andAUC_(0-∞) of sulindac were all included within the range of 80.00% to125.00%. The results of this comparison indicate that bioequivalencecriteria were met when Treatment 3 and Treatment 4 were administeredunder fasted conditions and demonstrate that the co-administration ofindividual tablets containing eflornithine or sulindac did not influencethe bioavailability of sulindac when administered alone.

TABLE 21 Summary of the Statistical Analysis of Sulindac in Treatment 3vs. Treatment 4 INTRA- GEOMETRIC LSMEANS * 90% CONFIDENCE SUBJECTTreatment-3 Treatment-4 RATIO LIMITS (%) PARAMETER C.V. (%) (n = 12) (n= 12) (%) LOWER UPPER C_(max) 24.6 4888.5 4704.2 103.92 87.56 123.32AUC_(0-T) 11.9 11063.6 10530.9 105.06 96.63 114.22 AUC_(0-∞) 13.612743.6 11834.3 107.68 93.31 124.27 * units are ng/mL for C_(max) and ng· h/mL for AUC_(0-T) and AUC_(0-∞) **n = 7 for AUC_(0-∞)

Sulindac Comparison between Treatment 1 and Treatment 4: Thepharmacokinetic results demonstrate that the geometric LSmean ratios andthe corresponding 90% confidence intervals (90 CI) of C_(max),AUC_(0-T), and AUC_(0-∞) of sulindac were not all included within therange of 80.00% to 125.00%. The lower bound of the 90 CI of C_(max) wasbelow the 80.00% limit. Since the ratios are within the 80.00% to125.00% range for all PK parameters, the intra-subject variability couldaccount for the lower bound of C_(max) being outside the BE range. Theresults obtained for this comparison demonstrate that the sample sizeused in this pilot study was not sufficient to demonstratebioequivalence of sulindac bioavailability from the co-formulated tabletand the co-administration of individual tablets containing eflornithineor sulindac.

TABLE 22 Summary of the Statistical Analysis of Sulindac in Treatment 1vs. Treatment 4 INTRA- GEOMETRIC LSMEANS * 90% CONFIDENCE SUBJECTTreatment-1 Treatment-4 RATIO LIMITS (%) PARAMETER C.V. (%) (n = 12) (n= 12) (%) LOWER UPPER C_(max) 24.6 4353.6 4704.2 92.55 77.98 109.83AUC_(0-T) 11.9 10746.4 10530.9 102.05 93.86 110.94 AUC_(0-∞) 13.612029.4 11834.3 101.65 88.09 117.30 * units are ng/mL for C_(max) and ng· h/mL for AUC_(0-T) and AUC_(0-∞) **n = 8 for AUC_(0-∞)

Based on the data, the intra-subject variation, that incorporates thevariability between all comparisons, is about 24.6% for C_(max) andabout 12% for AUC_(0-T). Statistically, given that the expectedTreatment 1 to Treatment 4 ratio of geometric LSmeans felt within 92.5and 107.5%, it is estimated that the number of subjects to meet the80.00 to 125.00% bioequivalence range with a statistical a priori powerof at least 80% would be about 36 for a future pivotal study. Theinclusion of 40 subjects should be sufficient to account for thepossibility of drop-outs and variations around the estimatedintra-subject CV.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to the methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A composition comprising a fixed dose combination in a single dosageunit of (a) about 375 mg of eflornithine hydrochloride monohydrate and(b) about 75 mg of sulindac, wherein the composition further comprisesmagnesium stearate. 2-3. (canceled)
 4. The composition of claim 1,wherein the eflornithine hydrochloride monohydrate is a racemic mixtureof its two enantiomers. 5-9. (canceled)
 10. The composition of claim 4,wherein the amount of eflornithine hydrochloride monohydrate racemate is375 mg. 11-19. (canceled)
 20. The composition of claim 1, wherein theamount of sulindac is 75 mg. 21-25. (canceled)
 26. The composition ofclaim 1, further comprising an excipient.
 27. The composition of claim26, wherein the excipient is starch, colloidal silicon dioxide, orsilicified microcrystalline cellulose.
 28. The composition of claim 26,wherein the excipient is colloidal silicon dioxide.
 29. The compositionof claim 28, wherein the composition further comprises a secondexcipient.
 30. The composition of claim 29, wherein the second excipientis silicified microcrystalline cellulose. 31-35. (canceled)
 36. Thecomposition of claim 1, wherein the amount of magnesium stearate is fromabout 1 to about 1.5 weight percent.
 37. (canceled)
 38. The compositionof claim 36, wherein the amount of magnesium stearate is about 1.5weight percent.
 39. The composition according to of claim 1, wherein thecomposition is in the form of a capsule, tablet, mini-tablet, granule,pellet, solution, gel, cream, foam, or patch.
 40. The composition ofclaim 39, wherein the composition is in the form of a tablet. 41.(canceled)
 42. The composition of claim 40, wherein the weight of thetablet is from about 675 mg to about 725 mg.
 43. The composition ofclaim 42, wherein the weight of the tablet is about 700 mg.
 44. Thecomposition of claim 40, wherein the tablet further comprises a coating.45-48. (canceled)
 49. The composition of claim 44, wherein the coatingcomprises hydroxypropyl methylcellulose, titanium dioxide, polyethyleneglycol, and iron oxide yellow.
 50. (canceled)
 51. The composition ofclaim 44, wherein the amount of coating is from about 2 to about 4weight percent. 52-56. (canceled)
 57. The composition of claim 44,wherein the weight of the tablet is from about 700 mg to about 725 mg.58. The composition of claim 57, wherein the weight of the tablet isabout 721 mg.
 59. A method of preventing and/or treating familialadenomatous polyposis (FAP) in a patient in need thereof, comprisingadministering to the patient a composition comprising a fixed dosecombination in a single dosage unit of (a) about 375 mg eflornithinehydrochloride monohydrate and (b) about 75 mg of sulindac, wherein thecomposition further comprises magnesium stearate. 60-94. (canceled)